Figure 1 ( A ) Representative micrographs of human skin sections stained with hematoxylin - eosin ( H & E ) ( left ) and anti - IL - 38 antibody ( right ) from normal patients ( n = 11 ) and tumors of cSCC patients ( n = 13 ) . Scale bars represent 100 μm . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . ( B ) Relative expression of IL - 38 in human normal tissues ( n = 9 ) and cSCC ( n = 18 ) were analyzed using Geo Datasets ( GSE98767 ) . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . ( C and D ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 32 weeks to induce skin tumors . ( C ) Representative micrographs of mouse normal skin ( n = 6 ) and tumor ( n = 6 ) sections stained with anti - IL - 38 antibody . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . Scale bars represent 100 μm . ( D ) Relative expression levels of Il - 38 in normal skin ( n = 5 ) and tumors ( n = 5 ) of mice were quantified using qPCR . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . ( F ) Representative western blot bands indicating IL - 38 in mouse normal skin ( n = 3 ) and DMBA / TPA - induced tumors ( n = 3 ) . The graph shows the quantification of mean IL - 38 expression in tissues . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. (B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.(F) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


Figure 2 ( B ) Representative photographs of Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA for 32 weeks . White triangles indicate the tumors on the back of mice . ( C Average tumor number ( C ) per mouse from Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA . . Error bars represent the mean ± SD ( C ) All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test (

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice. (C Average tumor number (C) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C


Figure 3 ( A ) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f ( n = 5 ) mice after treated with DMBA for 24h . Red triangles indicate the γH2AX + positive cells . Scale bars represent 100 μm . The graph shows the number of γH2AX + cells per high - powered field . Data information : Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of γH2AX+ cells per high-powered field. Data information: Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


Figure 4 ( A The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( A ) Representative micrograph sections stained with hematoxylin - eosin ( H & E ) from the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f mice ( n = 5 ) Scale bars represent 100 μm . The graph shows average epidermal thickness . B ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( B ) Representative micrograph sections stained with anti - Ki67 antibody from the skin of Il - 38f / f ( n = 6 ) and K14Cre / + - Il - 38f / f mice ( n = 6 ) . Scale bars represent 100 μm . The graph shows average numbers of Ki67 + cells per high - powered field . ( G and H ) Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) after overexpression ( G ) or knockdown ( H ) of IL - 38 . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression . ( I and J ) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL - 38 overexpression ( I ) or knockdown ( J ) . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph sections stained with hematoxylin-eosin (H&E) from the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal thickness.B) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (B) Representative micrograph sections stained with anti-Ki67 antibody from the skin of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.(G and H) Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) after overexpression (G) or knockdown (H) of IL-38. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test. NC, Negative control; LV, Lentivirus; Oe, Overexpression.(I and J) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL-38 overexpression (I) or knockdown (J). NC, Negative control; LV, Lentivirus; Oe, Overexpression.


Figure 5 ( A and B ) Pull - down assay of IL - 38 ( 1 - 152 aa ) ( A ) or IL - 38 ( 20 - 152 aa ) ( B ) with IL - 1Rrp2 - Fc , IL - 1RAPL1 - Fc , IL1RAcp - Fc , and IgG - Fc fusion proteins . ( C ) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector ( left ) or an expression vector encoding IL - 1Rrp2 ( right ) were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) or IL - 36β ( 200 ng / ml ) at different times . ( D ) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 at different concentrations . ( F ) Cell extracts prepared from 293T cells transfected with HA - tagged full - length or deletion mutants of IL - 1Rrp2 , were probed using anti - HA or anti - β - actin antibodies . Oe , overexpression . ( G ) Cell extracts prepared from 293T cells transfected with full - length or deletion mutants of IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) at different times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A and B) Pull-down assay of IL-38 (1-152 aa) (A) or IL-38 (20-152 aa) (B) with IL-1Rrp2-Fc, IL-1RAPL1-Fc, IL1RAcp-Fc, and IgG-Fc fusion proteins.(C) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector (left) or an expression vector encoding IL-1Rrp2 (right) were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) or IL-36β (200 ng/ml) at different times.(D) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 at different concentrations.(F) Cell extracts prepared from 293T cells transfected with HA-tagged full-length or deletion mutants of IL-1Rrp2, were probed using anti-HA or anti-β-actin antibodies. Oe, overexpression.(G) Cell extracts prepared from 293T cells transfected with full-length or deletion mutants of IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) at different times.


Figure 6Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after IL - 1Rrp2 interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after IL-1Rrp2 interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Figure 7Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after JNK interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after JNK interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Figure 1B - J : Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs . Antibodies against Tuj1 , Gfap and Olig2 were used to recognize neurons , astrocytes and oligodendrocytes . In D , G and J the percentage of cells positive ( mean ± SEM ) for each marker was counted over the total of DAPI positive cells ( 100 % ) . No difference between genotypes was highlighted ( Two - way ANOVA ) ; the variation through time resulted significant for the levels of Tuj1 and Gfap ( Two - way ANOVA : F ( 1 , 16 ) = 9 . 542 , p - value < 0 . 05 and F ( 1 , 10 ) = 68 . 95 , p - value < 0 . 0001 respectively ) . Sample size : n ≥ 3 wells obtained from two independent preparations ; for each well 10 random fields were acquired . Scale bars : 30 µm . P : Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14 . For each gene Two - way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) . Values are represented as mean ± SEM . Sample size : ≥ 5 samples obtained from two independent preparations .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm.P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.


Figure 2H - K : Raster plots ( H and I ) and their quantitation under basal conditions and upon 100µM 4 - AP exposure . The magnitude of the response after stimulation is expressed either as fold change ( J ) or as the averaged maximal values reached after 4 - AP exposure ( K ) . Dotted line in J represents untreated controls ( set to 1 ) . Values are represented as mean ± SEM . Student ' s t - test , * : p - value < 0 . 05 . Sample size n = 8 wells deriving from three independent preparations . L : Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points . The binary mask is representative of a neuron used to study morphology . Scale bars : 30 µm . M - Q : Each graph represents a different morphological parameter analysed at selected time point . Two - way ANOVA indicated both a significant time effect ( ( F 4 , 20 ) = 282 , 5 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 104 , 5 and p < 0 , 0001 for the total dendritic length ; ( F 4 , 20 ) = 58 , 51 and p < 0 , 0001 for the maximal dendritic length ; ( F 3 , 22 ) = 38 , 32 and p < 0 , 0001 for polarization ) and genotype effect ( ( F 1 , 20 ) = 25 , 1 and p < 0 , 0001 for the number of intersections ; ( F 4 , 26 ) = 16 , 0 and p = 0 . 005 for the total dendritic length ; ( F 1 , 20 ) = 17 , 24 and p = 0 , 005 for the maximal dendritic length ; ( F 1 , 22 ) = 18 , 76 and p < 0 . 003 for polarization ) . Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point . The dotted line in panel P represents the cut - off above which a culture is defined polarized ( Horton et al , 2006 ) . Values are represented as mean ± SEM ; * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * * : p - value < 0 . 0001 . Sample size : n ≥ 3 wells deriving from three independent preparations ; for each well we measured at least 15 cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2H-K: Raster plots (H and I) and their quantitation under basal conditions and upon 100µM 4-AP exposure. The magnitude of the response after stimulation is expressed either as fold change (J) or as the averaged maximal values reached after 4-AP exposure (K). Dotted line in J represents untreated controls (set to 1). Values are represented as mean ± SEM. Student's t-test, *: p-value<0.05. Sample size n=8 wells deriving from three independent preparations.L: Wt and null neurons counterstained with an antibody against Tuj1 and DAPI at different time points. The binary mask is representative of a neuron used to study morphology. Scale bars: 30 µm. M-Q: Each graph represents a different morphological parameter analysed at selected time point. Two-way ANOVA indicated both a significant time effect ((F 4,20)=282,5 and p<0,0001 for the number of intersections; (F 4,26)=104,5 and p<0,0001 for the total dendritic length; (F 4,20)=58,51 and p<0,0001 for the maximal dendritic length; (F 3,22)=38,32 and p<0,0001 for polarization) and genotype effect ((F 1,20)=25,1 and p<0,0001 for the number of intersections; (F 4,26)=16,0 and p=0.005 for the total dendritic length; (F 1,20)=17,24 and p=0,005 for the maximal dendritic length; (F 1,22)=18,76 and p<0.003 for polarization). Bonferroni multiple comparison test was used to assess difference between wt and null neurons at each analysed time point. The dotted line in panel P represents the cut-off above which a culture is defined polarized (Horton et al, 2006). Values are represented as mean ± SEM; *: p-value<0.05; **: p-value<0.01; ****: p-value<0.0001. Sample size: n≥3 wells deriving from three independent preparations; for each well we measured at least 15 cells.


Figure 3B , C : Five morphological parameters were measured in untreated and treated null neurons at DIV14 . One - way ANOVA indicated a significant treatment effect for the maximal dendritic length ( F = 6 , 306 ; p = 0 , 0039 ) , total dendritic length ( F = 4 , 880 ; p = 0 , 0126 ) , number of intersections ( F = 10 , 70 ; p = 0 , 0003 ) and soma area ( F = 7 , 721 ; p = 0 , 0015 ) . Tukey ' s multiple comparison test was used to assess differences between null neurons and early and late treated neurons . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 . The variation of each of the selected parameters is summarized in the radar plot ( C ) . Sample size ( panels B , C ) : n ≥ 18 wells for ctrl group and n ≥ 10 wells for the treatment groups deriving from three independent preparations ; for each well we measured at least 15 cells . Each point is representative of a single well . Values are represented as mean ± SEM . Bar graphs represent the expression values ( mean ± SEM ) of 27 genes , selected for their downregulation in null samples at DIV 14 ( p - value < 0 , 07 ; see Appendix Figure S2 ) , after CX546 administration in the two different time windows ( early in G and late in H ) . One - way ANOVA followed by Dunnett ' s multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls ( set to 1 , red dotted line ) . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 , * * * : p - value < 0 . 001 , * * * * : p - value < 0 . 0001 . Sample size : n ≥ 8 wells deriving from two independent preparations . Each point is representative of a single well .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B,C: Five morphological parameters were measured in untreated and treated null neurons at DIV14. One-way ANOVA indicated a significant treatment effect for the maximal dendritic length (F=6,306;p=0,0039), total dendritic length (F=4,880;p=0,0126), number of intersections (F=10,70; p=0,0003) and soma area (F=7,721;p=0,0015). Tukey's multiple comparison test was used to assess differences between null neurons and early and late treated neurons. *: p-value<0.05; **: p-value<0.01; ***: p-value<0.001. The variation of each of the selected parameters is summarized in the radar plot (C). Sample size (panels B,C): n≥18 wells for ctrl group and n≥10 wells for the treatment groups deriving from three independent preparations; for each well we measured at least 15 cells. Each point is representative of a single well. Values are represented as mean ± SEM.Bar graphs represent the expression values (mean ± SEM) of 27 genes, selected for their downregulation in null samples at DIV 14 (p-value <0,07; see Appendix Figure S2), after CX546 administration in the two different time windows (early in G and late in H). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare the expression of each gene between Mecp2 null early and late treated samples and their corresponding untreated controls (set to 1, red dotted line). *: p-value<0.05; **: p-value<0.01, ***: p-value<0.001, ****: p-value<0.0001. Sample size: n≥8 wells deriving from two independent preparations. Each point is representative of a single well.


Figure 4F , G : Violin plots represent the average values of MQAE intensity for early ( F ) or late ( G ) treated null neurons and their relative controls . One - way ANOVA followed by Tukey ' s multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons . * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 . The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles . Sample size : n ≥ 300 cells from five different embryos per genotype . I , J : In I One - way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons ( set to 100 ) , null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4 . * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 ( * : p - value < 0 . 05 ; * * : p - value < 0 . 01 ; * * * : p - value < 0 . 001 ) ; # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine ( # : p - value < 0 . 05 ; # # : p - value < 0 . 01 ) . Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 ( expressed as 2 - ∆ ct ) . Box plot shows the median ( central band ) , the lower and upper quartiles ( boxes ) and the max and min values ( whiskers ) from at least 3 wells obtained from four different embryos . One - way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones . * * : p - value < 0 . 01 . Sample size : n ≥ 3 wells from four different embryos .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4F,G: Violin plots represent the average values of MQAE intensity for early (F) or late (G) treated null neurons and their relative controls. One-way ANOVA followed by Tukey's multiple comparison test was used to compare null control neurons and null treated neurons to wt neurons. ***: p<0.001; ****: p <0.0001. The central line of the violin plot is the median while the two dotted lines represent the upper and lower quartiles. Sample size: n≥300 cells from five different embryos per genotype.I,J: In I One-way ANOVA was used to compare the expression of 9 neuronal genes between Mecp2 null primary neurons (set to 100), null neurons treated with CX546 from DIV1 to DIV4 and null neurons treated with CX546 and Nifedipine from DIV1 to DIV4. * refers to differences between Mecp2 null untreated neurons and Mecp2 null neurons treated with CX546 (*: p-value<0.05; **: p-value<0.01; ***: p-value<0.001); # refers to differences between Mecp2 null neurons treated with CX546 and null neurons treated with both CX546 and Nifedipine (#: p-value<0.05; ##: p-value<0.01). Panel J shows the ratio between the expression level of Kcc2 and Nkcc1 (expressed as 2-∆ct). Box plot shows the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 3 wells obtained from four different embryos. One-way ANOVA was used to compare the effects of the different treatments on null neurons compared to the untreated ones. **: p-value<0.01. Sample size: n≥3 wells from four different embryos.


Figure 5B : Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals . Sample size : wt = 11 , ko = 16 , ko treated with CX546 = 11 . Gehan - Breslow - Wilcoxon test was used to compare the two groups . * * : p - value < 0 . 01 . F , G : Line graph ( F ) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials . Two - way ANOVA indicated a significant effect of time ( F ( 2 , 21 ) = 5 , 490 and p = 0 , 0121 ) and genotype ( F ( 2 , 33 ) = 16 , 33 and p < 0 . 0001 ) . Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546 . * : p - value < 0 . 05 ; * * : p - value < 0 . 01 . The scatter plot ( G ) shows latency to fall ( in seconds ) on the accelerating rotarod in the last trial . One - way ANOVA was used to compare ko mice and ko treated mice to the wt group . The Tukey ' s multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine ; * * : p - value < 0 . 01 . Values are represented as average ± SEM . Sample size : n = 6 wt treated with vehicle and n = 14 ko ( 6 treated with vehicle and 8 treated with CX546 ) . Each dot represents a single animal .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B: Kaplan Mayer test shows an improved lifespan of Mecp2 null CX546 treated animals compared to Mecp2 null control animals. Sample size: wt=11, ko=16, ko treated with CX546=11. Gehan-Breslow-Wilcoxon test was used to compare the two groups. **: p-value<0.01.F,G: Line graph (F) shows mice motor learning assessed on the accelerating rotarod during three consecutive trials. Two-way ANOVA indicated a significant effect of time (F(2,21)=5,490 and p=0,0121) and genotype (F(2,33)=16,33 and p<0.0001). Bonferroni multiple comparison test highlighted a significant increase in the latency to fall between trial 1 and trial 3 only for null mice treated with CX546. *: p-value<0.05; **: p-value<0.01. The scatter plot (G) shows latency to fall (in seconds) on the accelerating rotarod in the last trial. One-way ANOVA was used to compare ko mice and ko treated mice to the wt group. The Tukey's multiple comparison test highlighted a significant reduction in the latency to fall between wt and ko but not between wt and ko treated with Ampakine; **: p-value<0.01. Values are represented as average ± SEM. Sample size: n=6 wt treated with vehicle and n=14 ko (6 treated with vehicle and 8 treated with CX546). Each dot represents a single animal.


Figure 1A , B After grown in nutrient rich medium to log phase , wild - type ( WT ) cells were treated with rapamycin ( 10 μM ) or subject to nitrogen starvation ( SD - N ) for 3 h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . The cycloheximide ( CHX , 100 μg / mL , ) or thiolutin ( 10 μg / mL ) was simultaneously used to block protein synthesis or gene transcription . Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected . Yeast cells were treated with thiolutin ( 10 μg / mL ) together with rapamycin or nitrogen starvation in different order for 3 h , 6h and 16 h and degradation of autophagic marker GFP - Atg8 was detected .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, B After grown in nutrient rich medium to log phase, wild-type (WT) cells were treated with rapamycin (10 μM) or subject to nitrogen starvation (SD-N) for 3 h and 16 h and degradation of autophagic marker GFP-Atg8 was detected. The cycloheximide (CHX, 100 μg/mL,) or thiolutin (10 μg/mL) was simultaneously used to block protein synthesis or gene transcription.Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.Yeast cells were treated with thiolutin (10 μg/mL) together with rapamycin or nitrogen starvation in different order for 3 h, 6h and 16 h and degradation of autophagic marker GFP-Atg8 was detected.


Figure 2B N - terminally GFP - tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1 , 4 and 16 h starvation . Yeast cells with ATG1 deletion were used as positive controls . H Vacuole localization and degradation of endocytosis substrate GFP - Sna3 in WT , atg1 ∆ and rpb9 ∆ cells . Scale bars : 5 μm . I Carboxypeptidases Y ( Cpy1 ) , a vacuolar - resident hydrolases , was analyzed for its transporting to vacuoles by the secretory pathway in WT , atg1 ∆ and rpb9 ∆ cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B N-terminally GFP-tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4 and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls.H Vacuole localization and degradation of endocytosis substrate GFP-Sna3 in WT, atg1∆ and rpb9∆ cells. Scale bars: 5 μm.I Carboxypeptidases Y (Cpy1), a vacuolar-resident hydrolases, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ and rpb9∆ cells.


Figure 3E - I Overexpression of ATG1 could partially restore autophagy in rpb9 ∆ cells . ATG1 , ATG9 , ATG13 , ATG17 and ATG5 were overexpressed in rpb9 ∆ yeast cells , GFP - 50Q was checked for its autophagic degradation by GFP processing assays after 1 , 4 and 16 h starvation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3E-I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1, ATG9, ATG13, ATG17 and ATG5 were overexpressed in rpb9∆ yeast cells, GFP-50Q was checked for its autophagic degradation by GFP processing assays after 1, 4 and 16 h starvation.


Figure 4B Indicated Rpb9 truncates and mutants were expressed in rpb9 ∆ cells and autophagic degradation of GFP - 50Q was checked by GFP - processing assays .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP-50Q was checked by GFP-processing assays.


Figure 5E Purified GST - Gcn4 and His - Rpb9 proteins from E . coli cells were mixed and GST pulldown assays were performed . F - G Neither overexpression of Gcn4 in rpb9 ∆ yeast cells nor overexpression of Rpb9 in gcn4 ∆ yeast cells can restore autophagy . Gcn4 or Rpb9 were overexpressed in rpb9 ∆ or gcn4 ∆ yeast cells respectively and autophagic degradation of GFP - 50Q was detected .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5E Purified GST-Gcn4 and His-Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed.F-G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP-50Q was detected.


Figure 6C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9 ∆ yeast cells . Indicated Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected . D N - terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9 ∆ yeast cells . Indicated truncates of Rpb9 orthologs were expressed in rpb9 ∆ yeast cells and autophagic degradation of GFP - 50Q was detected .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.D N-terminal Zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP-50Q was detected.


Figure 1A . Glioma tumour micro arrays ( grade II - IV gliomas ) were stained to visualise MDGI ( red ) . Samples from the epileptic brain served as a negative control . Scale bar 100 µm . B . Overall survival of grade II and III glioma patients ( n = 76 ) was significantly better when none / low MDGI ( blue line ) was detected compared to patients with moderate / high MDGI ( red line ) expression ( P = 0 . 007 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method . C . Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal . In the TCGA GBMLGG dataset ( n = 667 ) , patients with low MDGI expression ( green line ; n = 333 , events = 101 , median = 67 , 5 ) show significantly better survival than patients with high MDGI ( red line ; n = 334 , events = 138 , median = 41 . 5 ) expression ( P = 2e - 04 ) . HR = 0 . 62 ( 0 . 48 - 0 . 8 ) . The cumulative survival rates were estimated by using the Kaplan - Meier method . D . In glioblastoma samples , highest MDGI expression was observed in the mesenchymal subtype ( M ) compared to the classical ( Cl ) or pro - neural ( PN ) ones . However , it did not reach the statistical significance . GlioVis data portal , TCGA GBM dataset , n = 156 . Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction . E . MDGI expression was high in the leading edge of the tumour ( LE ) and in infiltrating tumour cells ( IT ) in the IVY _ Gap dataset ( n = 122 ) where the gene expression profile in different anatomical structures of glioblastomas was analysed . LE , leading edge ; IT , infiltrating tumour cells ; CT , cellular tumour ( cells within the tumour mass ) ; PC , pseudopalisading cells ; MP , microvascular proliferation . Numbers in the graph depict the P values : Pairwise t - test with corrections for multiple testing , p - values with Bonferroni correction . F . Western blot analysis shows MDGI expression in human glioma cells including patient - derived spheroids ( S24 , ZH161 , ZH305 , BT3 , BT11 , BT12 , BT13 ) , patient - derived adherent cells ( BT5 and BT5R ) , and long - term cell lines ( U87MG , LN229 , LN308 ) . Numbers above the lanes show the relative expression of MDGI compared to the levels of β - tubulin that served as loading control in each cell line . Representative Western blot image of 3 separate experiments is shown . G . High MDGI and HIF - 1α expression in the patient - derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum ( FBS ) for seven days . Numbers show the relative expression of MDGI and HIF - 1α compared to the levels of β - tubulin . Expression in the serum - free medium was set as 1 . Representative Western blot image of 2 separate experiments is shown . H . MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia ( 1 % O2 ) or normoxia ( 21 % O2 ) . Numbers show the relative MDGI expression compared to the levels of β - tubulin . Expression under normoxia was set as 1 . Representative Western blot image of 3 separate experiments is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Glioma tumour micro arrays (grade II-IV gliomas) were stained to visualise MDGI (red). Samples from the epileptic brain served as a negative control. Scale bar 100 µm.B. Overall survival of grade II and III glioma patients (n = 76) was significantly better when none/low MDGI (blue line) was detected compared to patients with moderate/high MDGI (red line) expression (P = 0.007). The cumulative survival rates were estimated by using the Kaplan-Meier method.C. Analysis of the association of MDGI mRNA expression with glioma patient survival using the GlioVis data portal. In the TCGA GBMLGG dataset (n = 667), patients with low MDGI expression (green line; n = 333, events = 101, median = 67,5) show significantly better survival than patients with high MDGI (red line; n = 334, events = 138, median = 41.5) expression (P = 2e-04). HR = 0.62 (0.48 - 0.8). The cumulative survival rates were estimated by using the Kaplan-Meier method.D. In glioblastoma samples, highest MDGI expression was observed in the mesenchymal subtype (M) compared to the classical (Cl) or pro-neural (PN) ones. However, it did not reach the statistical significance. GlioVis data portal, TCGA GBM dataset, n = 156. Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.E. MDGI expression was high in the leading edge of the tumour (LE) and in infiltrating tumour cells (IT) in the IVY_Gap dataset (n = 122) where the gene expression profile in different anatomical structures of glioblastomas was analysed. LE, leading edge; IT, infiltrating tumour cells; CT, cellular tumour (cells within the tumour mass); PC, pseudopalisading cells; MP, microvascular proliferation. Numbers in the graph depict the P values: Pairwise t-test with corrections for multiple testing, p-values with Bonferroni correction.F. Western blot analysis shows MDGI expression in human glioma cells including patient-derived spheroids (S24, ZH161, ZH305, BT3, BT11, BT12, BT13), patient-derived adherent cells (BT5 and BT5R), and long-term cell lines (U87MG, LN229, LN308). Numbers above the lanes show the relative expression of MDGI compared to the levels of β-tubulin that served as loading control in each cell line. Representative Western blot image of 3 separate experiments is shown.G. High MDGI and HIF-1α expression in the patient-derived glioblastoma spheroids decreased significantly when cells were cultured in medium containing 10 % of serum (FBS) for seven days. Numbers show the relative expression of MDGI and HIF-1α compared to the levels of β-tubulin. Expression in the serum-free medium was set as 1. Representative Western blot image of 2 separate experiments is shown.H. MDGI expression in adherent glioma cell lines and in glioma cell spheroids after 24h culture under hypoxia (1 % O2) or normoxia (21 % O2). Numbers show the relative MDGI expression compared to the levels of β-tubulin. Expression under normoxia was set as 1. Representative Western blot image of 3 separate experiments is shown.


Figure 2A , B Representative whole coronal section micrographs of murine brain injected with GFP ( A ) or MDGI - GFP ( B ) expressing U87MG glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in C and D ) , Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in D point to the angiotropic tumours . Scale bar : 50 µm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in E and F ) , and CD31 ( red in E and F ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . Arrows in F point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm . G . Quantification of the number of U87MG secondary tumours ( diameter > 300 µm ) detected in the whole brain ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . H . Quantification of the number of invasive U87MG cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) ( GFP : n = 5 , MDGI - GFP : n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . I , J Representative micrographs of whole murine brain coronal sections injected with GFP ( I ) or GFP - MDGI ( J ) expressing LN229 glioma cells . Xenografted human cells were visualised by using anti - human vimentin ( hVim ) antibodies ( red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for human vimentin ( hVim red in K and L ) Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in L point to the angiotropic tumours . Scale bar : 50 µm . Micrographs of consecutive ( separating distance : 9 µm ) brain sections stained for human glioma cells using antibodies specific for MDGI ( green in M and N ) , and CD31 ( red in M and N ) . Dashed lines separate the primary tumour mass and the normal brain . Nuclei were visualised by using DAPI ( blue ) . ) . Arrows in N point to the MDGI - expressing angiotropic tumour cells . Scale bar : 50 µm . O . Quantification of the number of invasive LN229 cells detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . P . Quantification of the number of invasive LN229 cells that grew next to brain blood vessels ( angiotropic co - opting tumour cells , diameter < 300 µm ) detected in the brain ( GFP : n = 10 , MDGI - GFP : n = 10 ) . Data are represented as mean ± SEM . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Scale bar: 50 µm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L) Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Scale bar: 50 µm.Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.


Figure 3A . Transmitted light micrographs of MDGI - silenced ( shMDGI1 and shMDGI2 ) and control ( Scr ) patient - derived BT12 and BT13 spheroids 11 days after gene silencing . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm . B . Methylcellulose assay demonstrating the ability for self - renewal of control ( Scr ) and MDGI - silenced ( shMDGI1 and shMDGI2 ) patient - derived BT12 and BT13 cells after three weeks of culture . Representative images of at least 3 independent experiments are shown . Scale bar : 200 μm . C . Quantification of the colony size in methylcellulose of MDGI - silenced ( sh1 and sh2 ) cells relative to the controls ( Scr ) that is set as 100 ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . D . MTT - proliferation assay of control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 and BT13 cells . The values of each cell line were normalized to the values at day 1 marked with a dashed line ( n = 3 ) . Data are represented as mean ± SD . * * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . E , F Representative whole coronal section micrographs of murine brain injected intracranially with control ( BT12 Scr , n = 5 ) and MDGI - silenced ( BT12 sh1 , n = 5 ) BT12 cells . After four weeks animals were sacrificed , the brains were excised and prepared for immunohistochemistry . Control ( Scr ) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells ( single cell invasion ) that eventually co - opted existing blood vessels ( angiotropism ) and formed secondary tumours ( upper panels ) . No tumour growth was observed when MDGI - silenced cells were implanted ( lower panels ) . Glioma cells were visualised by using anti - human vimentin ( hVim red ) and blood vessels using anti - podocalyxin antibodies ( green ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm ( E ) and 50 µm ( F ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).


Figure 4A . Binding of Annexin V Alexa Fluor 488 to control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 cells was measured by flow cytometric analyses five days after silencing . The analysis was performed by gating the cells into two populations : P1 ( small , granular ) and P2 ( normal - sized cells ) ( 30 000 analysed events / cell line , n = 3 ) . B . Geometric mean fluorescence ( FL - 1 ) relative to the controls ( Scr ) that was set as 1 in P1 and P2 in MDGI - silenced ( shMDGI1 ) cells . Data are represented as mean ± SD . * P < 0 . 05 , two - tailed , nonparametric Mann - Whitney ' s U - test . C , D Representative micrographs of non - treated control BT12 cells ( C ) and cells treated with 2 mM LLOMe for 4 h ( D ) before staining with the anti - galectin - 1 antibodies ( LGALS1 ) ( green ) . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . E , F Representative micrographs of LGALS1 - stained ( green ) BT12 control ( E ) and MDGI - silenced cells ( F ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . G , H Representative micrographs of LGALS1 - stained ( green ) BT13 control ( G ) and MDGI - silenced cells ( H ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . I . Percentage of LGALS1 - positive puncta staining in the control ( Scr ) and MDGI - silenced ( sh1 ) cells . In total 0 . 4 - 1 . 2x104 LGALS1 - stained cells were analysed from 50 mm2 coverslip areas ( n = 6 ) . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . J . Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining ( n = 100 ) in control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . K . Cytoplasmic cathepsin B activity in the control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 ; * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . L . Number of live cells in MDGI - silenced BT12 and BT13 cells incubated without ( sh1 ) or with ( sh1 + K777 ) the pan - cathepsin inhibitor ( K777 ) after 1 , 3 and 5 days of incubation . Data shows the percentage of live cells compared to the control ( Scr ) cells set as 100 % . Data are represented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.I. Percentage of LGALS1-positive puncta staining in the control (Scr) and MDGI-silenced (sh1) cells. In total 0.4-1.2x104 LGALS1-stained cells were analysed from 50 mm2 coverslip areas (n = 6). Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.J. Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining (n = 100) in control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.K. Cytoplasmic cathepsin B activity in the control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01; **P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


Figure 5A . Proportions ( mol % ) of saturated ( Sat ) , monounsaturated ( Mono ) , diunsaturated ( Di ; total of 2 double bonds in the acyl chains ) , and polyunsaturated ( Poly ; total of 3 - 7 double bonds in the acyl chains ) lipid species from the two main phospholipid classes ( phosphatidylcholine PC and phosphatidylethanolamine PE ) of the lysosomal fractions of the BT12 and BT13 control ( Scr ) and MDGI - silenced ( shMDGI ) cells . A representative graph of two independent experiments is shown . B . Fold change of the main PC and PE species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) cells ( Log2 , by mol % data ) . Abbreviations : [ Lipid class ] [ total number of acyl chain carbons ] : [ total number of double bonds in the chains ] . A representative heatmap of two independent experiments is shown . C . Profiles of ceramide ( Cer ) species ( mol % ) in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . Abbreviations : Cer [ number of acyl chain carbons ] : [ number of double bonds in the acyl chain ] ; all principal species containing a sphingosine chain . A representative graph of two independent experiments is shown . D . Ratio of the 24 : 1 to 24 : 0 ( mol % . mol % ) Cer species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . A representative graph of two independent experiments is shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.


Figure 6A . Measurement of the BT12 , BT13 , and ZH305 glioblastoma cell viability using the MTT - assay at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability . B . Measurement of the viability of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and embryonic kidney ( HEK293T ) cells at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability . C . Representative micrographs of BT12 , BT13 , and ZH305 glioblastoma cells treated with 1 µg / ml of clemastine for 24 h and stained with anti - galectin - 1 ( LGALS1 ) antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 20 µm . D . Representative micrographs of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and kidney cells ( HEK293T ) treated with 1 µg / ml clemastine for 24 h and stained with anti - LGALS1 antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


Figure 7A . Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells ) , and daily treated with saline vehicle ( Ve ) or 50 mg / kg clemastine ( Cle , 100 mg / kg single dose administered at day 1 ) for 12 days starting at day 15 after tumour implantation . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , ZH305 ( Ve n = 10 , Cle n = 10 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Human glioma cells were visualised with an anti - human vimentin ( hVim , red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . B , C Quantification of the number of the invading single BT12 wt glioblastoma cells ( B ) and distance of the invaded cells from the primary tumour ( C ) of vehicle ( black bars , Ve ) and clemastine - treated ( white bars , Cle ) animals ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . D . Quantification of the number of secondary tumours ( diameter > 300 µm ) in the murine brain for the indicated treatments and tumour models . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . E . Quantification of the number of angiotropic BT12 cells that have co - opted existing blood vessels in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . G . Representative micrographs of consecutive brain sections ( separating distance : 9 µm ) showing xenografts treated with vehicle or clemastine and labelled with the anti - human vimentin ( red in left panels ) and TUNEL ( red in right panels ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 50 µm . H . Kaplan - Meier survival curve of the BT12 xenografted animals treated with vehicle ( green line , n = 15 ) and clemastine ( black line , n = 12 ) . Survival of the animals has been measured and pooled from two independent experiments . P = 0 . 044 , Mantel - Cox Log - rank test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.


Figure 1 ( A , B ) Isolated epidermal LCs ( A , n = 4 ) or isolated vaginal LCs ( B , n = 5 ) were stimulated O / N with a variety of vaginal microbiota and subsequently exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) . Epidermal LC were stimulated with L . crispatus ( LC ) , M . elsdenii ( ME ) , and P . timonensis ( PT ) whereas vaginal LCs were additionally stimulated with L . iners ( LI ) , G . vaginalis ( GV ) , and A . vaginae ( AV ) . After five days , HIV - 1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( C ) Isolated epidermal LCs ( n = 3 ) were stimulated O / N with B . fragilis ( BF ) , B . thetaiotaomicron ( BT ) , P . amnii ( PA ) , P . bivia ( PB ) , P . copri ( PC ) , and P . timonensis ( PT ) , followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) infection for 5 days and HIV - 1 p24 determination by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( D ) Vaginal epithelium explants ( n = 8 ) were exposed to vaginal microbiota as described for isolated vaginal LCs . HIV - 1 p24 in emigrated LCs was determined by flow cytometry . Data information : symbols represent independent donors . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A, B) Isolated epidermal LCs (A, n = 4) or isolated vaginal LCs (B, n = 5) were stimulated O/N with a variety of vaginal microbiota and subsequently exposed to HIV-1 (SF162; MOI 0.5). Epidermal LC were stimulated with L. crispatus (LC), M. elsdenii (ME), and P. timonensis (PT) whereas vaginal LCs were additionally stimulated with L. iners (LI), G. vaginalis (GV), and A. vaginae (AV). After five days, HIV-1 p24 in LCs was determined by an intracellular staining for CD1a and p24 by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(C) Isolated epidermal LCs (n = 3) were stimulated O/N with B. fragilis (BF), B. thetaiotaomicron (BT), P. amnii (PA), P. bivia (PB), P. copri (PC), and P. timonensis (PT), followed by HIV-1 (SF162; MOI 0.5) infection for 5 days and HIV-1 p24 determination by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(D) Vaginal epithelium explants (n = 8) were exposed to vaginal microbiota as described for isolated vaginal LCs. HIV-1 p24 in emigrated LCs was determined by flow cytometry. Data information: symbols represent independent donors. Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Figure 2 ( A - D ) Isolated epidermal LCs ( A , n = 2 ) , isolated vaginal LCs ( B - C , both n = 3 ) , or vaginal explants ( D , n = 5 ) were stimulated O / N with P . timonensis ( PT ) and exposed to HIV - 1 ( SF162 ; MOI 0 . 5 ) for five days in presence or absence of HIV - 1 replication inhibitors ( Inh ) zidovudine ( AZT ) , tenofovir ( TFV ) , iamivudine ( 3TC ) or indinavir ( IDV ) . HIV - 1 levels were determined by intracellular staining for HIV - 1 p24 using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 . ( G ) Isolated epidermal LCs ( n = 3 ) were treated with T20 , Maraviroc ( MAR ) , neutralizing antibodies against CD4 ( αCD4 ) and isotype control ( Iso ) , followed by O / N stimulation with P . timonensis ( PT ) . Next , LCs were exposed to HIV - 1 for five days ( SF162 ; MOI 0 . 5 ) . Intracellular HIV - 1 p24 levels were determined using flow cytometry . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A-D) Isolated epidermal LCs (A, n = 2), isolated vaginal LCs (B-C, both n = 3), or vaginal explants (D, n = 5) were stimulated O/N with P. timonensis (PT) and exposed to HIV-1 (SF162; MOI 0.5) for five days in presence or absence of HIV-1 replication inhibitors (Inh) zidovudine (AZT), tenofovir (TFV), iamivudine (3TC) or indinavir (IDV). HIV-1 levels were determined by intracellular staining for HIV-1 p24 using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.(G) Isolated epidermal LCs (n = 3) were treated with T20, Maraviroc (MAR), neutralizing antibodies against CD4 (αCD4) and isotype control (Iso), followed by O/N stimulation with P. timonensis (PT). Next, LCs were exposed to HIV-1 for five days (SF162; MOI 0.5). Intracellular HIV-1 p24 levels were determined using flow cytometry. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, and ***p <0.001.


Figure 3Immature vaginal ( vLC ) and epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) and activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured and activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . Representative plots of vaginal ( A ) or epidermal ( B ) donors and combined experiments ( MFI expression relative to untreated condition ) of CD80 , CD86 and CCR7 surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . Immature vaginal ( vLC ) and epidermal LCs ( eLC ) were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , or P . timonensis ( PT ) and activation phenotype was determined by flow cytometry . Same donor emigrated epidermal LCs ( mLC ) served as a matured and activated control . Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue ( N . D . ) . CD80 ( C , n = 3 ) , CD86 ( D n = 3 ) , and CCR7 ( E , n = 3 ) surface expression are shown . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . Ex vivo skin explants were stimulated O / N with L . crispatus ( LC ) , M . elsdenii ( ME ) , P . timonensis ( PT ) or poly ( I : C ) and at day 3 post - inoculation , emigrated LCs were collected , washed and activation phenotype was determined by flow cytometry . Cells were analysed for CD1a expression and the absolute number of CD1a - positive cells , i . e . LCs , migrated from the epidermis was determined using counting beads , the graph shows the migration relative to the untreated condition ( F , n = 5 ) . CD1a - positive cells , i . e . LCs , were analysed using flow cytometry and the graphs show combined experiments ( MFI expression relative to untreated condition ) of CD80 ( G , n = 5 ) and CD86 ( H n = 5 ) surface expression . Data information : symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). Representative plots of vaginal (A) or epidermal (B) donors and combined experiments (MFI expression relative to untreated condition) of CD80 , CD86 and CCR7 surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.Immature vaginal (vLC) and epidermal LCs (eLC) were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), or P. timonensis (PT) and activation phenotype was determined by flow cytometry. Same donor emigrated epidermal LCs (mLC) served as a matured and activated control. Same donor data of emigrated vaginal LCs could not be obtained due to limited amount of tissue (N.D.). CD80 (C, n = 3), CD86 (D n = 3), and CCR7 (E, n = 3) surface expression are shown. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.Ex vivo skin explants were stimulated O/N with L. crispatus (LC), M. elsdenii (ME), P. timonensis (PT) or poly(I:C) and at day 3 post-inoculation, emigrated LCs were collected, washed and activation phenotype was determined by flow cytometry. Cells were analysed for CD1a expression and the absolute number of CD1a-positive cells, i.e. LCs, migrated from the epidermis was determined using counting beads, the graph shows the migration relative to the untreated condition (F, n = 5). CD1a-positive cells, i.e. LCs, were analysed using flow cytometry and the graphs show combined experiments (MFI expression relative to untreated condition) of CD80 (G, n = 5) and CD86 (H n = 5) surface expression. Data information: symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, *p  < 0.05, **p  < 0.01, ***p <0.001, and ****p < 0.0001.


Figure 4Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure . ( A ) Intracellular HIV - 1 detection was determined by confocal microscopy ( Hoechst in blue , HIV - 1 p24 in green ) , scale bar represents 5 µm ( representative donor ) . ( B ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by a 4 - hour exposure to HIV - 1 . HIV - 1 p24 was determined by ELISA after trypsin treatment and lysis of LCs ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( C ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 ( SF162 ; MOI 0 . 5 ) exposure for 3 days . After 3 days , LCs were treated with PBS or trypsin and HIV - 1 p24 was determined by flow cytometry ( n = 3 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( D , E ) Isolated vaginal LCs were stimulated O / N with P . timonensis ( PT ) followed by infection with VSV - g - BlaM - Vpr fusion ( positive control ) and NL4 . 3Bal - BlaM - Vpr fusion . Figure shows representative plots ( D ) and pooled data ( E , n = 3 ) of viral fusion as determined by β - lactamase - Vpr ( BlaM - Vpr ) activity . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( F ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by HIV - 1 exposure ( SF162 ; MOI 0 . 5 ) in presence or absence of anti - langerin ( 10E2 ) or mannan ( MAN ) . HIV - 1 p24 content was determined by ELISA after trypsin - treatment and subsequent lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 . ( G ) Isolated epidermal LCs were stimulated O / N with P . timonensis ( PT ) followed by treatment with Rapamycin ( µM ) and exposure to HIV - 1 ( SF162 ) . After 3 days HIV - 1 p24 was measured by ELISA after trypsin treatment and lysis of LCs ( n = 2 ) . Data information : Symbols represent independent donors ( mean of duplicates ) . Data are mean ± SD . Two - tailed t - test , * * p < 0 . 01 , * * * p < 0 . 001 , and * * * * p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure. (A) Intracellular HIV-1 detection was determined by confocal microscopy (Hoechst in blue, HIV-1 p24 in green), scale bar represents 5 µm (representative donor).(B) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by a 4-hour exposure to HIV-1. HIV-1 p24 was determined by ELISA after trypsin treatment and lysis of LCs (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(C) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 (SF162; MOI 0.5) exposure for 3 days. After 3 days, LCs were treated with PBS or trypsin and HIV-1 p24 was determined by flow cytometry (n = 3). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(D, E) Isolated vaginal LCs were stimulated O/N with P. timonensis (PT) followed by infection with VSV-g-BlaM-Vpr fusion (positive control) and NL4.3Bal-BlaM-Vpr fusion. Figure shows representative plots (D) and pooled data (E, n = 3) of viral fusion as determined by β-lactamase-Vpr (BlaM-Vpr) activity. Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(F) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by HIV-1 exposure (SF162; MOI 0.5) in presence or absence of anti-langerin (10E2) or mannan (MAN). HIV-1 p24 content was determined by ELISA after trypsin-treatment and subsequent lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.(G) Isolated epidermal LCs were stimulated O/N with P. timonensis (PT) followed by treatment with Rapamycin (µM) and exposure to HIV-1 (SF162). After 3 days HIV-1 p24 was measured by ELISA after trypsin treatment and lysis of LCs (n = 2). Data information: Symbols represent independent donors (mean of duplicates). Data are mean ± SD. Two-tailed t-test, **p  < 0.01, ***p <0.001, and ****p < 0.0001.