Figure 8A . Continuous osmotic delivery of miR - 10b inhibitor markedly reduces tumor burden . The osmotic pumps , loaded with lipid nanoparticles formulated with 2 ' - O - MOE - POmiR - 10b inhibitor or non - targeting control infused 2 μg of the oligonucleotides per day introtumorally , over 13 days . Tumors growth was monitored by the WBI , and the left panels illustrate tumor imaging of representative animals at the end of treatment . The bars represent average signal ratios for each group at day 13 ( end of treatment ) , relative to day 2 ( treatment initiation ) . B . Representative immunostaining of tumors for PCNA proliferation marker . C . Representative immunostaining of the tumors for KI67 proliferation marker . D . Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis . E . Tumor cell invasion was examined by fluorescence microscopy for mCherry - positive cells migrating through the tumor border . F . Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti - miR - 10b treatment . No significant effect on invasion of intracranial GBM8 was observed .

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 B-PANEL_START 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 B-PANEL_START 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

Figure 8A. Continuous osmotic delivery of miR-10b inhibitor markedly reduces tumor burden. The osmotic pumps, loaded with lipid nanoparticles formulated with 2'-O-MOE-POmiR-10b inhibitor or non-targeting control infused 2 μg of the oligonucleotides per day introtumorally, over 13 days. Tumors growth was monitored by the WBI, and the left panels illustrate tumor imaging of representative animals at the end of treatment. The bars represent average signal ratios for each group at day 13 (end of treatment), relative to day 2 (treatment initiation).B. Representative immunostaining of tumors for PCNA proliferation marker.C. Representative immunostaining of the tumors for KI67 proliferation marker.D. Representative tumor immunostaining for cleaved Caspase 3 as a marker of apoptosis.E. Tumor cell invasion was examined by fluorescence microscopy for mCherry-positive cells migrating through the tumor border.F. Quantitative immunostainings analysis indicates that proliferation and apoptosis markers are affected by anti-miR-10b treatment. No significant effect on invasion of intracranial GBM8 was observed.


Figure 9A , B . miR - 10b inhibition decreases GL261 cellviability . Cellviability was measured at days 3 - 7 after transfection with miR - 10b inhibitor , non - targeting control , or Lipofectamine 2000 alone ( mock ) . A . Phase contrast photographs of GL261 cultures at day 6 post - transfection . B . Growth curves of cultured GL261 cells , based on the viability assay . C . InVivo - jet PEI - formulated 2 ' - O - MOE - PS / PO miR - 10b inhibitor or non - targeting control were infused to orthotopic GL261 tumors by osmotic pumps , starting at day 6 after cell implantation . The uptake of ASOs was confirmed by IHC for PS - containing oligonucleotides ( green ) . GL261 tumor cells expressing M - Cherry are red . D . Osmotic delivery of miR - 10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice . Micephotographs show tumor imaging in representative animals at day 3 after pump implantation , and average signals in photons per second are indicated . Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment . E . The efficacy of miR - 10b inhibition in intracranial tumors was assessed by qRT - PCR analysis of the resected tumor tissues , with miR - 10b expression levels normalized to miR - 125b . F . qRT - PCR analysis demonstrates that miR - 10b target p21 was de - repressed in GL261 tumors upon miR - 10b inhibition . mRNA expression levels were normalized to GAPDH .

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 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 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 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 9A, B. miR-10b inhibition decreases GL261 cellviability. Cellviability was measured at days 3-7 after transfection with miR-10b inhibitor, non-targeting control, or Lipofectamine 2000 alone (mock).A. Phase contrast photographs of GL261 cultures at day 6 post-transfection.B. Growth curves of cultured GL261 cells, based on the viability assay.C. InVivo-jet PEI - formulated 2'-O-MOE-PS/PO miR-10b inhibitor or non-targeting control were infused to orthotopic GL261 tumors by osmotic pumps, starting at day 6 after cell implantation. The uptake of ASOs was confirmed by IHC for PS-containing oligonucleotides (green). GL261 tumor cells expressing M-Cherry are red.D. Osmotic delivery of miR-10b inhibitor markedly reduces GL261tumorgrowth in immunocompetent Black 6 Albino mice. Micephotographs show tumor imaging in representative animals at day 3 after pump implantation, and average signals in photons per second are indicated. Tumorgrowth rates were calculated as ratios of the signals at day 3 of the treatment to day 1 prior to initiation of the treatment.E. The efficacy of miR-10b inhibition in intracranial tumors was assessed by qRT-PCR analysis of the resected tumor tissues, with miR-10b expression levels normalized to miR-125b.F. qRT-PCR analysis demonstrates that miR-10b target p21 was de-repressed in GL261 tumors upon miR-10b inhibition. mRNA expression levels were normalized to GAPDH.


Figure 1 ( B ) Western blot analysis of lysates and media from wt and mutant N9 cells ( N9 wt / mu ) using antibody anti - murine TREM2 ( clone 5F4 ) , which is raised against the murine TREM2 extracellular domain . sTREM2 , soluble TREM2 * Indicate unspecific bands . Anti - calnexin antibody was used as loading control . ( C ) Phagocytosis of 1 µM HiLyte ™ Fluor 488 Aβ1 - 42 ( fAβ42 ) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non - binding antibody 6687 . Cytochalasin D ( CytoD , 10 mM ) was used as control to verify phagocytic uptake . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 61 , genotype P < 0 . 0001 , treatment P = 0 . 0001 ; post hoc tests wt vs . mu for the following conditions : fAβ42 P = 0 . 0043 , fAβ42 - 2D8 P = 0 . 0436 ) . ( D ) Western blot of BMDM derived from wt and Trem2 knockout ( ko ) animals using antibody 5F4 . ( E ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8 , or the non - binding control antibody 6687 . ( n = 3 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0005 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 P = 0 . 0021 , fAβ42 - 2D8 1 µg / ml P < 0 . 0001 , fAβ42 - 2D8 5 µg / ml P < 0 . 0001 , fAβ42 - 2D8 10 µg / ml P < 0 . 0001 , fAβ42 / 6687 10 µg / ml P = 0 . 0007 ) ( F ) Quantification of relative fAβ42uptake to lowest antibody concentration used . ( G ) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0223 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 1 µg / ml P = 0 . 0391 , fAβ42 - mAb11 5 µg / ml P = 0 . 0069 , fAβ42 - mAb11 10 µg / ml P < 0 . 0001 , fAβ42 - mAb11 20 µg / ml P = 0 . 0001 , fAβ42 - mAb11 50 µg / ml P < 0 . 0001 ) . ( H ) Quantification of relative fAβ42 uptake to lowest antibody concentration used . ( I ) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2 - deficient BMDM . Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 ( 10 µg / ml ) . ( J ) Western blot of primary microglia from wt or Trem2 ko animals using antibody 5F4 . ( K ) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11 , or an isotype control antibody ( IC ) . ( n = 5 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 4797 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; post hoc tests wt vs . ko for the following conditions : fAβ42 - mAb11 5 µg / ml P = 0 . 0449 , fAβ42 - mAb11 10 µg / ml P = 0 . 0370 , fAβ42 - mAb11 20 µg / ml P = 0 . 0299 , fAβ42 - mAb11 50 µg / ml P = 0 . 0120 ) . ( L ) Quantification of relative fAβ42uptake to lowest antibody concentration used .

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

Figure 1(B) Western blot analysis of lysates and media from wt and mutant N9 cells (N9 wt /mu) using antibody anti-murine TREM2 (clone 5F4), which is raised against the murine TREM2 extracellular domain. sTREM2, soluble TREM2* Indicate unspecific bands. Anti-calnexin antibody was used as loading control.(C) Phagocytosis of 1 µM HiLyte™ Fluor 488 Aβ1-42 (fAβ42) by N9 wt and N9 mu in the presence or absence of antibody 2D8 or the non-binding antibody 6687. Cytochalasin D (CytoD, 10 mM) was used as control to verify phagocytic uptake. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.61, genotype P<0.0001, treatment P=0.0001; post hoc tests wt vs. mu for the following conditions: fAβ42 P=0.0043, fAβ42-2D8 P=0.0436).(D) Western blot of BMDM derived from wt and Trem2 knockout (ko) animals using antibody 5F4.(E) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of 2D8, or the non-binding control antibody 6687. (n = 3, +/- SEM; 2 way ANOVA, interaction P=0.0005, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42 P=0.0021, fAβ42-2D8 1 µg/ml P<0.0001, fAβ42-2D8 5 µg/ml P<0.0001, fAβ42-2D8 10 µg/ml P<0.0001, fAβ42/6687 10 µg/ml P=0.0007)(F) Quantification of relative fAβ42uptake to lowest antibody concentration used.(G) Phagocytosis of fAβ42 by BMDM from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0223, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 1 µg/ml P=0.0391, fAβ42-mAb11 5 µg/ml P=0.0069, fAβ42-mAb11 10 µg/ml P<0.0001, fAβ42-mAb11 20 µg/ml P=0.0001, fAβ42-mAb11 50 µg/ml P<0.0001).(H) Quantification of relative fAβ42 uptake to lowest antibody concentration used.(I) Recombinant mousesTREM2 does not rescue fAβ42 uptake in Trem2-deficient BMDM. Increasing amounts of sTREM2 were added to the media of wt or Trem2 ko BMDM in the presence or absence of mAb11 (10 µg/ml).(J) Western blot of primary microglia from wt or Trem2 ko animals using antibody 5F4.(K) Phagocytosis of fAβ42 by primary microglia from wt and Trem2 ko animals in the presence or absence of mAb11, or an isotype control antibody (IC). (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.4797, genotype P<0.0001, treatment P<0.0001; post hoc tests wt vs. ko for the following conditions: fAβ42-mAb11 5 µg/ml P=0.0449, fAβ42-mAb11 10 µg/ml P=0.0370, fAβ42-mAb11 20 µg/ml P=0.0299, fAβ42-mAb11 50 µg/ml P=0.0120).(L) Quantification of relative fAβ42uptake to lowest antibody concentration used.


Figure 2 ( A ) Representative histograms for Fcγ - receptors - PE ( FcγR - PE ) expression levels as used for quantification . Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR ( I , IIB / III and IV ) . ( B ) Relative quantification of cell surface levels of FcγR molecules . Absolute number of cell surface FcγR - PE molecules was determined by the BD QuantiBRITE ® method ( see methods section for details ) and normalized to expression levels of the respective wt control . ( n = 4 , + / - SEM , t test , two - tailed ; wt vs . ko : FcγRI - PE P = 0 . 0008 , FcγRIIB / III - PE P = 0 . 0033 , FcγRIV - PE P = 0 . 7001 ) . ( C ) mRNA levels of FcγR are increased in Trem2 ko BMDM . Fold changes of the respective FcγR ( I , IIB , III , IV ) mRNA levels in Trem2 ko BMDM were determined by quantitative real - time PCR . ( n = 6 , + / - SEM ; one - sample t test , two - tailed ; wt vs . ko : FcγRI P = 0 . 0118 , FcγRIIB P = 0 . 0006 , FcγRIII P = 0 . 0004 , FcγRIV P = 0 . 0284 ) ( D ) Phosphorylated Syk ( P - Syk ) and total Syk ( T - Syk ) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone , together with antibody 2D8 ( Aβ - 2D8 ) , or an isotype control ( Aβ - IC ) . Actin was used as a loading control . ( E ) Quantification of P - Syk normalized to T - Syk . ( n = 4 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0490 , genotype P = 0 . 0898 , treatment P < 0 . 0001 . Bonferroni - corrected pair - wise post hoc tests , * * P = 0 . 0075 vs . wt ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative histograms for Fcγ-receptors-PE (FcγR-PE) expression levels as used for quantification. Stacked histograms for log PE fluorescence intensity of wt and Trem2 ko BMDM are shown for the respective FcγR (I, IIB/III and IV).(B) Relative quantification of cell surface levels of FcγR molecules. Absolute number of cell surface FcγR-PE molecules was determined by the BD QuantiBRITE® method (see methods section for details) and normalized to expression levels of the respective wt control. (n=4, +/- SEM, t test, two-tailed; wt vs. ko: FcγRI-PE P=0.0008, FcγRIIB/III-PE P=0.0033, FcγRIV-PE P=0.7001).(C) mRNA levels of FcγR are increased in Trem2 ko BMDM. Fold changes of the respective FcγR (I, IIB, III, IV) mRNA levels in Trem2 ko BMDM were determined by quantitative real-time PCR. (n=6, +/- SEM; one-sample t test, two-tailed; wt vs. ko: FcγRI P=0.0118, FcγRIIB P=0.0006, FcγRIII P=0.0004, FcγRIV P=0.0284)(D) Phosphorylated Syk (P-Syk) and total Syk (T-Syk) levels were determined by western blotting in lysates from wt and Trem2 ko BMDM after 1 h treatment with Aβ alone, together with antibody 2D8 (Aβ-2D8), or an isotype control (Aβ-IC). Actin was used as a loading control.(E) Quantification of P-Syk normalized to T-Syk. (n = 4, +/- SEM; 2 way ANOVA, interaction P=0.0490, genotype P=0.0898, treatment P<0.0001. Bonferroni-corrected pair-wise post hoc tests, ** P =0.0075 vs. wt).


Figure 3 ( B ) mAb11 but not the IC co - localized with methoxy - X04 . BMDM ( C ) or primary microglia ( D ) were cultured on cryosections pre - incubated with mAb11 ( 1 µg / ml ) . After 24 h , sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy - X04 staining to visualize Aβ . Note that both cell types internalize Aβ into intracellular vesicles ( right panels show enlargement of insets ) . Scale bar : 10µm .

O O B-PANEL_START 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

Figure 3(B) mAb11 but not the IC co-localized with methoxy-X04.BMDM (C) or primary microglia (D) were cultured on cryosections pre-incubated with mAb11 (1 µg/ml). After 24 h, sections were processed for immunostaining using antibody against CD68 to identify myeloid cells and methoxy-X04 staining to visualize Aβ. Note that both cell types internalize Aβ into intracellular vesicles (right panels show enlargement of insets). Scale bar: 10µm.


Figure 4 ( A ) BMDM from wt or Trem2 ko mice were cultured on APP / PS1micebrain cryosections incubated with or without mAb11 ( 1 µg / ml ) or an isotype control ( IC ; 1 µg / ml ) for 24 h . Sections were then probed with methoxy - X04 . Scale bar , 500 µm . ( B ) The amyloid plaque load was quantified from the entire sagittal section . Sections incubated with medium ( no cell ) were set as baseline . ( n = 6 , + / - SEM ; 2 way ANOVA , interaction P < 0 . 0001 , genotype P < 0 . 0001 , treatment P < 0 . 0001 ; Tukey ' s multiple comparisons tests ; wt vs . ko for the following conditions : no antibody P = 0 . 0304 , IC P = 0 . 0049 , mAb11 P = 0 . 0212 ; wt : IC vs . wt : mAb11 P = 0 . 0008 ; ko : IC vs . ko : mAb11 P = 0 . 0001 ) . ( C , D ) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections . ( n = 4 , + / - SEM ; t - test ; n . s . , nonsignificant , P = 0 . 5004 ) . Scale bar , 200 µm . ( E ) Aβ was extracted by urea buffer from replicate slices of the experiment shown in ( A ) and total Aβ was identified by western blotting .

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 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

Figure 4(A) BMDM from wt or Trem2 ko mice were cultured on APP/PS1micebrain cryosections incubated with or without mAb11 (1 µg/ml) or an isotype control (IC; 1 µg/ml) for 24 h. Sections were then probed with methoxy-X04. Scale bar, 500 µm.(B) The amyloid plaque load was quantified from the entire sagittal section. Sections incubated with medium (no cell) were set as baseline. (n = 6, +/- SEM; 2 way ANOVA, interaction P<0.0001, genotype P<0.0001, treatment P<0.0001; Tukey's multiple comparisons tests; wt vs. ko for the following conditions: no antibody P=0.0304, IC P=0.0049, mAb11 P=0.0212; wt : IC vs. wt : mAb11 P=0.0008; ko : IC vs. ko : mAb11 P=0.0001).(C, D) Equal numbers of wt and Trem2 ko BMDM were added and cell numbers were analyzed after termination of experiments by quantifying the CD68 positive cells on top of the sections. (n = 4, +/-SEM; t-test; n.s., nonsignificant, P=0.5004). Scale bar, 200 µm.(E) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.


Figure 5 ( A ) Cryosections from unfixed brain of 6 - months old APP / PS1mice were pre - incubated with increasing concentrations of mAb11 ( 0 . 001 , 0 . 01 , 0 . 1 , 1 , 5 µg / ml ) . BMDM from wt or Trem2 ko mice were added for 24 h . Sections were stained with methoxy - X04 . Scale bar , 500 µm . ( B ) Methoxy - X04 signals were quantified from the entire sagittal section . ( n = 5 , + / - SEM ; 2 way ANOVA , interaction P = 0 . 0082 , genotype P < 0 . 0001 , treatment P < 0 . 0001 . Fisher ' s LSD post hoc comparisons ; * show statistics between wt and ko under the same experimental condition . # in black show wt compares to no antibody stimulation ; # in grey show ko compares to no antibody stimulation ; wt vs . ko for the following conditions : no antibody P = 0 . 0053 , mAb11 0 . 001 µg / ml P = 0 . 0003 , mAb11 0 . 01 µg / ml P < 0 . 0001 , mAb11 0 . 1 µg / ml P = 0 . 0001 , mAb11 1 µg / ml P = 0 . 0007 , mAb11 5 µg / ml P = 0 . 0011 ; Following conditions compare to wt / no antibody : wt / mAb11 0 . 01 µg / ml P = 0 . 0166 , wt / mAb11 0 . 1 µg / ml P = 0 . 0002 , wt / mAb11 1 µg / ml P < 0 . 0001 , wt / mAb11 5 µg / ml P < 0 . 0001 ; Following conditions compare to ko / no antibody : ko / mAb11 0 . 1 µg / ml P = 0 . 0099 , ko / mAb11 1 µg / ml P < 0 . 0001 , ko / mAb11 5 µg / ml 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.


Figure 1Dynamic fluorescence patterns according to cell density in 8 × GTIIC - EmGFP 293T cells . EmGFP intensity in LATS2 knockdown , or forced expression of a constitutively active form of YAP ( YAP - S127A ) . Scale bars indicate 100 μm . Scatter plots of mean frequency of each shRNA per gene . Average of v HOXA4 is indicated as a red dot . Augmented 8 × GTIIC - luciferase reporter activity by loss of HOXA4 . ShSAV1 was used as an experimental control . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4Quantitative real - time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression

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Figure 1Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression vector


Figure 2Quantitative real - time PCR analysis of CTGF , CYR61 , and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4 . ShLATS1 / 2 and shYAP were used as an experimental control . * P < 0 . 05 , * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction . 8 × GTIIC - luciferase reporter assays in HEK 293T cells transfected HOXA4 and / or shLATS1 / 2 . * * P < 0 . 01 vs shCtrl , by two - way ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with overexpression of HOXA4 and / or constitutive active form of YAP ( YAP - 5SA ) . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with LacZ , by ANOVA with Sidak ' s correction . Quantitative real - time PCR analysis of HOXA4 , YAP , CTGF , and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and / or overexpression of YAP - 5SA . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with shCtrl , by ANOVA with Sidak ' s correction .

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Figure 2Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.


Figure 3Association of HOXA4 with TEAD1 indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc - TEAD1Association of HOXA4 but not YAP indicated by co - IP experiments . HEK 293T cells were co - transfected with FLAG - HOXA4 and Myc - YAP . Interaction between endogenous TEAD1 and HOXA4 by co - IP experiments in HEK 293T cells . The interaction between endogenous TEAD1 and YAP was used as a positive control . Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1 . Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4 . Arrowheads indicate IgG heavy chain . Arrows indicate TEAD1 ( a . a . 1 - 426 ) . Requirement of TEAD1 ( a . a . 30 - 135 ) for its interaction with HOXA4 via its homeodomain ( a . a . 216 - 274 ) . Failure of TEAD1 ( a . a . 30 - 102 ) , TEA domain alone , to interact with HOXA4 .

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Figure 3Association of HOXA4 with TEAD1 indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1Association of HOXA4 but not YAP indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-YAP.Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.


Figure 4ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4 . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments . Competitive co - IP assay and densitometry showing significantly reduced TEAD - YAP interaction by overexpression of HOXA4 in a dose - dependent manner in HEK 293T cells . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments .

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Figure 4ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Figure 5Expression of Hoxa4 in various mouse tissues using qRT - PCR . Expression of glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) mRNA was used as an internal control . WAT ; white adipose tissue , BAT ; brown adipose tissue , Skm ; skeletal muscle . Quantitative real - time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF - BB stimulation for 24 hours . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Quantitative real - time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP / TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 . Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 . Quantitative real - time PCR analysis of YAP / TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP . The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - tailed Student ' s t - test . Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction . BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction . Proliferation assay in VSMCs transduced with the indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cells were collected and counted at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . BrdU incorporation assays in VSMCs transduced with the indicated genes . The percentage of BrdU positive cells in each group is shown . Nine to twelve images were randomly acquired and analyzed . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Figure 5Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Figure 6Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1 / 3 / 4 . Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Proliferation assay in VSMCs transduced with indicated genes . Virus - transfected human VSMCs were plated at equal density in growth medium , and then cell viability was analyzed by Cell Counting Kit - 8 at indicated days at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments . Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co - IP experiments in human VSMCs with knockdown of HOXA4 . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± SEM of three independent experiments . ChIP assays of CTGF , CYR61 and α - SMA promoters using anti - FLAG - tag , anti - Myc - tag , anti - TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc - tagged YAP - 5SA and / or FLAG - tagged HOXA4 . * P < 0 . 05 , * * P < 0 . 01 vs FLAG - GFP , by unpaired two - tailed Student ' s t - test . Data are expressed as the means ± SEM of three independent experiments .

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Figure 6Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Figure 7Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) . Quantitative real - time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 . Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation ( n = 6 ) . Quantitative real - time PCR analysis of Yap / Tead target genes in the left carotid artery of 8 - week - old WT and Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) . Quantitative real - time PCR analysis of Yap / Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at the baseline was defined as 1 . 0 . Representative western blotting analysis and densitometry of non - ligated ( right ; R ) and ligated ( left ; L ) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation ( WT : n = 6 , Hoxa4 KO : n = 7 ) . * P < 0 . 05 , * * P < 0 . 01 , by two - way ANOVA with Sidak ' s correction . Representative images showing Elastica van Gieson staining ( G ) , and vessel , neointima and media area ( H ) , and neointima to media layer ratio ( I ) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation ( WT : n = 14 , Hoxa4 KO : n = 11 ) .

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Figure 7Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6).Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.Representative western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction.Representative images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).


Figure 1WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of CHIKV . ( E ) Viral tissue burdens were analyzed at 1 day post inoculation ( dpi ) by FFA . Mean ± SEM . WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of ONNV ( F ) Tissues and serum were collected at 1 dpi and analyzed by plaque assay . WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 103 PFU of RRV ( G ) . Tissues and serum were collected at 1 dpi and analyzed by plaque assay .

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Figure 1WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. (E) Viral tissue burdens were analyzed at 1 day post inoculation (dpi) by FFA. Mean ± SEM.WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of ONNV (F) Tissues and serum were collected at 1 dpi and analyzed by plaque assay.WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of RRV (G). Tissues and serum were collected at 1 dpi and analyzed by plaque assay.


Figure 2 ( A ) WT and Clec4F - DTR + C57BL / 6 mice were treated with diphtheria toxin ( DT ) either intraperitoneally ( i . p . ) 24 h prior to inoculation ( n = 10 , data pooled from two experiments , black dots ) or intravenously ( i . v . ) 48 and 24 h prior to inoculation ( n = 4 , one experiment , grey dots ) to deplete KCs and inoculated i . v . with 108 CHIKV particles . Viral genomes in the inoculum and serum at 45 minutes ( min ) - post inoculation were determined by RT - qPCR . Mean ± SD . Data are pooled from three experiments , n = 14 . Mann - Whitney test ; * * * * P < 0 . 0001 . ( C ) WT or CD169 - DTR + mice were treated i . p . with DT prior to i . v . inoculation of 108 CHIKV particles . Viral genomes were quantified as in ( A ) . Mean ± SD . Data are pooled from two experiments , n = 7 - 8 . Mann - Whitney test ; * * * P < 0 . 001 . ( D ) Livers from WT , Clec4F - DTR + or CD169 - DTR + mice treated i . p . with DT , or WT mice treated i . v . with PLL or CLL were analyzed by IHC to visualize F4 / 80 + macrophages ( brown ) . Two representative sections for each group are shown .

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 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 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

Figure 2(A) WT and Clec4F-DTR+ C57BL/6 mice were treated with diphtheria toxin (DT) either intraperitoneally (i.p.) 24 h prior to inoculation (n=10, data pooled from two experiments, black dots) or intravenously (i.v.) 48 and 24 h prior to inoculation (n=4, one experiment, grey dots) to deplete KCs and inoculated i.v. with 108 CHIKV particles. Viral genomes in the inoculum and serum at 45 minutes (min)-post inoculation were determined by RT-qPCR. Mean ± SD. Data are pooled from three experiments, n= 14. Mann-Whitney test; ****P < 0.0001.(C) WT or CD169-DTR+ mice were treated i.p. with DT prior to i.v. inoculation of 108 CHIKV particles. Viral genomes were quantified as in (A). Mean ± SD. Data are pooled from two experiments, n=7-8. Mann-Whitney test; ***P < 0.001.(D) Livers from WT, Clec4F-DTR+ or CD169-DTR+ mice treated i.p. with DT, or WT mice treated i.v. with PLL or CLL were analyzed by IHC to visualize F4/80+ macrophages (brown). Two representative sections for each group are shown.


Figure 3 ( A ) WT or Clec4F - DTR + C57BL / 6 mice were treated i . v . with DT prior to s . c . inoculation in the left rear footpad with 103 PFU of CHIKV . Infectious virus was quantified at 24 hpi by FFA . Mean ± SEM . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; P > 0 . 05 . ( B ) WT C57BL / 6 mice were treated i . v . with PLL or CLL 42 h prior to s . c . inoculation in the left rear footpad with 103 PFU of CHIKV . Infectious virus at 24 hpi was quantified by FFA . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; * * * 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 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

Figure 3(A) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus was quantified at 24 hpi by FFA. Mean ± SEM. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.(B) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.


Figure 4 ( B ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R . Viral genomes in the dLN and serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 10 . Two - way ANOVA with Bonferroni ' s multiple comparisons test ; * * * P < 0 . 001 , * * * * P < 0 . 0001 . ( C ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the left rear footpad with 108 particles of RRV . Viral genomes in the dLN and serum were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 - 9 . Mann - Whitney test ; * * * P < 0 . 001 , * * * * 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 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

Figure 4(B) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=10. Two-way ANOVA with Bonferroni's multiple comparisons test; ***P < 0.001, ****P < 0.0001.(C) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of RRV. Viral genomes in the dLN and serum were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8-9. Mann-Whitney test; ***P < 0.001, ****P < 0.0001.


Figure 5 ( A ) CD169 - DTR + mice were treated i . p . with PBS or DT prior to collection of the popliteal LN . Frozen LN sections were stained for CD169 + macrophages ( green ) , B220 + B cells ( blue ) or ERTR - 7 + stromal cells ( red ) . Image shown is representative of three biological replicates . Scale bars : 200 μm . ( B ) WT or CD169 - DTR + C57BL / 6 mice were treated i . p . with DT prior to s . c . inoculation in the left rear footpad with 108 particles of WT CHIKV . Viral genomes in the dLN and serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney test ; P > 0 . 05 .

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Figure 5(A) CD169-DTR+ mice were treated i.p. with PBS or DT prior to collection of the popliteal LN. Frozen LN sections were stained for CD169+ macrophages (green), B220+ B cells (blue) or ERTR-7+ stromal cells (red). Image shown is representative of three biological replicates. Scale bars: 200 μm.(B) WT or CD169-DTR+ C57BL/6 mice were treated i.p. with DT prior to s.c. inoculation in the left rear footpad with 108 particles of WT CHIKV. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.


Figure 6WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in the rear feet with 5 * 104 PFU of CHIKV - E2 - mCherry and the popliteal dLNs were collected at 2 hpi . ( A ) Frozen dLN sections were stained for Lyve - 1 + LECs ( white ) , MARCO ( green ) , B220 ( blue ) and mCherry + CHIKV particles ( red ) . Scale bars : 200 μm .

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Figure 6WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the rear feet with 5*104 PFU of CHIKV-E2-mCherry and the popliteal dLNs were collected at 2 hpi. (A) Frozen dLN sections were stained for Lyve-1+ LECs (white), MARCO (green), B220 (blue) and mCherry+ CHIKV particles (red). Scale bars: 200 μm.


Figure 7 ( H , I ) MARCO ( H ) and Mxra8 ( I ) expression is shown for MARCO + LECs for mock - infected cells and CHIKV - infected cells classified as either CHIKV - low or CHIKV - high . P values were calculated using a two - sided Wilcoxon rank - sum test with Bonferroni correction . In the boxplot , the central lines , the box limits , and the whiskers represent medians , the interquartile range ( IQR ) , and min / max values that are not outliers , respectively . Outliers are shown as points and include any values that are more than 1 . 5x IQR away from the box .

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Figure 7(H, I) MARCO (H) and Mxra8 (I) expression is shown for MARCO+ LECs for mock-infected cells and CHIKV-infected cells classified as either CHIKV-low or CHIKV-high. P values were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction. In the boxplot, the central lines, the box limits, and the whiskers represent medians, the interquartile range (IQR), and min/max values that are not outliers, respectively. Outliers are shown as points and include any values that are more than 1.5x IQR away from the box.


Figure 1 ( A , B ) Localization of oskar mRNA in wild - type ( A ) and in Tm1eg9 / Tm1eg9 ( B ) egg - chambers . ( C ) Kymograph of oskMS2 - GFP mRNPs ( cyan ) travelling along polarity marked MTs ( red , EB1 protein ) in an ex vivo ooplasmic preparation . White dashed arrow shows the growing plus tip of the MT . Scale bars represent 1 s and 1 µm , respectively . ( D ) Distribution of oskMS2 - GFP mRNP runs towards plus ( white ) and minus ends ( grey ) . Numbers within the bars indicate number of runs . P value of Chi2 test against wild - type is indicated above each bar . ( E , F ) oskar mRNA ( green ) and Oskar protein ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers expressing oskarMS2 ( 6x ) ( E ) or oskarMS2 ( 6x ) and Khc401 - MCP ( F ) . ( G ) oskar mRNA ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers rescued with EmGFP - Tm1 - I ( green ) . Scale bars represent 50 µm .

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Figure 1(A, B) Localization of oskar mRNA in wild-type (A) and in Tm1eg9/Tm1eg9 (B) egg-chambers.(C) Kymograph of oskMS2-GFP mRNPs (cyan) travelling along polarity marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 µm, respectively.(D) Distribution of oskMS2-GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate number of runs. P value of Chi2 test against wild-type is indicated above each bar.(E, F) oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc401-MCP (F).(G) oskar mRNA (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers rescued with EmGFP-Tm1-I (green). Scale bars represent 50 µm.


Figure 2 ( A - B " ) Colocalization of oskMS2 - mCherry ( red , A , B ) ( A ' , B ' ) with Khc - EGFP ( green , A ) ( A ' ) or with EmGFP - Tm1 - I ( green B ) ( B ' ) in ex vivo ooplasmic preparations . ( C ) Fraction of oskMS2 - MCP - mCherry mRNPs located non - randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations . MCP indicates MCP - EGFP which , like MCP - mCherry , can bind to MS2 loops . Staufen ( Stau ) is a dsRNA binding protein and bona fide partner of oskar mRNA ( St Johnston et al . , 1991 ; St Johnston et al . , 1992 ) . All values are significantly different from zero ( p < 10 - 3 , one sample t - test ) . ( D ) Fraction of oskMS2 - mCherry mRNPs co - localizing ( max . 200 nm ) non - randomly with Khc - EGFP particles in wild - type and in Tm1eg1 / Tm1eg9 ooplasms in presence of two ( white ) or one ( grey ) copy of endogenous Khc . ( C , D ) P values of two sample t - tests are indicated above the relevant bar pairs . Numbers indicate the number of particle clusters ( 160 mRNPs in each ) and the number of preparations ( in brackets ) analysed . Error bars represent 95 % confidence intervals .

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Figure 2(A-B") Colocalization of oskMS2-mCherry (red, A, B) (A', B') with Khc-EGFP (green, A) (A') or with EmGFP-Tm1-I (green B) (B') in ex vivo ooplasmic preparations.(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.


Figure 3 ( A , B ) Kymographs of oskMS2 - GFP mRNPs ( green ) associated with Khc - mKate2 ( A , red ) and mCherry - Tm1 - I / C ( B , red ) ex vivo . Arrows indicate motile RNPs in stable complex with Khc ( A ) or Tm1 - I / C ( B ) , the arrowheads point to non - motile oskMS2 - RNPs showing no obvious accumulation of the tagged protein . Note that mCherry - Tm1 - I was exposed twice as long as Khc - mKate to obtain comparable red fluorescence signals . Scale bars represent 1 µm and 1 second , respectively . ( C - E ) Relative Khc - mKate2 ( C ) , mCherry - Tm1 - I / C ( D ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots . ( C - E ) Relative GFP - Staufen ( E ) coverage of motile ( white ) and non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within the boxes indicate the number of trajectories and the number of ooplasms ( in brackets ) analysed . Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein ( for at least half of the duration of the trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U tests are indicated above the boxplots . ( F ) Example kymograph of Khc molecules ( red ) associating with an oskMS2 - GFP mRNP ( green ) before and during its run . White arrowheads indicate two association events , yellow arrow indicates the onset of motility . Scale bars represent 5 µm and 1 second , respectively . ( G ) Frequency of Khc - mKate2 appearance on motile ( white ) , before ( motility - primed , checked ) and after ( dotted ) the onset of motility , and non - motile ( grey ) oskMS2 - GFP trajectories . Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed . Indicated P values show results of pairwise Fisher ' s exact test . The Khc association frequency observed on RNPs before ( checked ) and during ( dotted ) their motility is not significantly different from wild - type motile RNP controls ( p > 0 . 01 ) .

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 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 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

Figure 3(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.(C-E) Relative GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.(F) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).


Figure 4 ( A ) Western blots of oskar mRNP components ( Staufen , Bruno and Y14 ) and motor associated proteins ( Khc , BicD , Dic ) co - immunoprecipitated with EmGFP - Tm1 - I from ovarian lysates . Protein marker bands and their molecular weight in kDa are indicated . ( B ) Signal developed with oligo ( dT ) 25 - TexasRed probes on GFP - Trap _ M beads binding EmGFP - Tm1 - I or FlagMycGFP after denaturing washes . 594 nm reflection marks bead boundaries . ( B ' ) Quantification of mean oligo ( dT ) signal measured on beads . ( C ) qRT - PCR of EmGFP - Tm1 - I and control Flag - Myc - GFP and bead alone ( w1118 ) bound oskar and gapdh RNAs after stringent washes ( mean ± s . e . m . , N = 3 ) . ( D ) Images of beads binding to EmGFP - Tm1 - I ( green ) and DIG labelled in vitro transcribed RNA fragments ( magenta ) . ( E , F ) Mean DIG - Cy5 fluorescence measured on beads capturing the RNA fragment , with or without UV cross - linking , indicated below the charts . P values of pairwise Mann - Whitney U tests are indicated . In panel F , none of the non - cross - linked samples differ significantly from the no RNA control ( p > 0 . 05 ) . Numbers below the plots indicate the number of beads analysed .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blots of oskar mRNP components (Staufen, Bruno and Y14) and motor associated proteins (Khc, BicD, Dic) co-immunoprecipitated with EmGFP-Tm1-I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.(B') Quantification of mean oligo(dT) signal measured on beads.(C) qRT-PCR of EmGFP-Tm1-I and control Flag-Myc-GFP and bead alone (w1118) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3).(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.


Figure 5 ( A , B , C ) Confocal image of a Tm1eg9 homozygous egg - chamber expressing EmGFP - Tm1 - I ( green ) and Khc - mKate2 ( magenta ) . oskar mRNA labelled with osk1 - 5 FIT probes ( Hovelmann et al . , 2014 ) is in yellow . ( B ' , C ' ) oskar mRNPs co - localizing with both EmGFP - Tm1 - I and Khc - mKate2 . Colors indicate the maximal co - localization distance ( C ' ) . Panels B - C ' represent the boxed regions in panel A . ( D , E ) Localization of Khc - mKate2 ( green ) and oskar mRNA ( magenta ) in wild - type and Tm1gs mutant nurse cells . ( F , G ) Fraction of oskar mRNPs co - localizing with Khc - mKate ( dark grey ) , EmGFP - Tm1 - I ( white ) , or both of these proteins ( light grey ) in the oocyte ( F ) or in the nurse cells ( G ) ( max . colocalization distance is 250 nm ) . None of the values are significantly different from each other ( one - way ANOVA , p > 10 - 3 ) . Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent ( see Fig EV3C and D ) . Significance of the observed co - localization values versus the expected values are shown . ( H ) Fraction of oskar mRNPs co - localizing with Khc - mKate in wild - type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled ( as indicated above the graph ) . P values of pair - wise t - tests are indicated . ( I ) Fraction of oskar mRNPs co - localizing with free mCherry in wild - type nurse cells used as negative control . The measured fraction ( ~ 1 . 6 % ) is significantly different from zero ( one sample t - test ) . All other measured co - localization values are significantly different form this negative control ( p < 0 . 001 , one - way ANOVA ) . ( F - I ) Numbers indicate the number of particle clusters ( 100 oskar mRNPs in each ) and the number of egg - chambers ( in brackets ) analysed . Error bars represent 95 % confidence intervals . All values ( F - I ) are significantly different from zero ( p < 10 - 3 , one sample 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 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

Figure 5(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.(B', C') oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C'). Panels B-C' represent the boxed regions in panel A.(D, E) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).


Figure 6 ( A - C ) Localization of EmGFP - Tm1 - I ( A ) oskar mRNA ( B ) and Khc ( C ) around the nurse cell nuclear envelope ( magenta , WGA staining ) . ( D - F ) Mean distribution profile of Khc ( D ) mKate2 - Tm1 - I , EmGFP - Tm1 - I ( E ) and oskar mRNA ( F ) around the NE of nurse cells ( marked with a dotted vertical line ) . Genotypes are indicated as follows : wild - type control with solid black line ( D - F ) , oskar RNA null with solid grey line ( D , E ) , Tm1eg9 / Tm1eg9 with dotted black line ( D , F ) Lines indicate mean and 95 % confidence intervals . We analysed the accumulation of mKate2 - Tm1 - I instead of EmGFP - Tm1 - I due to unexpected GFP expression from the oskar0 allele . ( G ) Khc accumulation around the NE . To calculate accumulation the signal intensity measured at the position of the peak observed in the wild - type control ( D , arrowhead , 356 ± 17 . 6 nm away from NE ) was divided by the signal intensity 2 s . d . away ( d , arrow , at 356 + 2 * 410 nm ) . P values of pairwise Mann - Whitney U tests against wild - type control or Tm1gs rescued with EmGFP - Tm1 - I are indicated above the boxplots . Numbers indicate the number of nuclei and the number of egg - chambers ( in brackets ) analysed . ( H - K ) Khc accumulation around the NE of nurse cells over - expressing EmGFP - Tm1 - I within Tm1eg9 / Tm1eg9 ( H ) , and oskar RNA null egg - chambers ( I ) expressing either the oskar 3 ' UTR ( J ) or oskar Δi ( 1 , 2 , 3 ) ( K ) .

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 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

Figure 6(A-C) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele.(G) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3' UTR (J) or oskar Δi(1,2,3) (K).


Figure 7 ( A - E ) Mean oskar mRNA distribution ( green , detected by conventional FISH ) within oocytes in which oskar mRNA is substituted by oskar Δi ( 1 , 2 , 3 ) mRNA ( A , B ) , oskar 3 ' UTR mRNA ( D ) and that ( in addition ) over - express EmGFP - Tm1 - I ( B , D and E ) . Wild - type control ( C ) and oocytes expressing Khc RNAi and EmGFP - Tm1 - I ( E ) . Magenta indicates standard deviation of the distribution of oskar mRNA . Scale bar is 10 % of total oocyte length ( C ) . ( F ) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte ( dotted horizontal line ) along the anteroposterior ( AP ) axis . Posterior pole is the top of the chart . P values of pairwise Mann - Whitney U tests are indicated . Numbers indicate number of oocytes analysed .

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

Figure 7(A-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3'UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.


Figure 1C Confirmatory screening performed as described above . Schematic distribution of compounds based on Z - score values of cells expressing polyGP - GFP and AUG - RFP ( above ) and on Z - score values of the fluorescent intensity of the two reporters per each compound ( below ) . Data are from four technical replicates , boxes are the Z - score mean value and whiskers represent ± SD . Baseline of Z - score = 0 indicates that Z - score is identical to the mean score . Asterisk ( * ) represents the selected compounds : 1 Forskolin ( FSK ) , 2 Erysolin ( ERY ) , 3 Geldanamycin ( GELD ) , 4 Helenin ( HLN ) and 5 Spironolactone ( SPL ) . Number sign ( # ) represents cellular stress inducers : # 1 Thapsigargin and # 2 Tunicamycin . D , E Dose - response analysis of ERY , HLN , SPL , FSK and GELD . Cells were co - transfected with AUG - RFP and polyGP - GFP plasmids and treated with two concentration ranges 0 . 5 , 1 , 5 and 10 µM ( D ) and 20 , 40 and 60 µM ( E ) for 24 h . CHX used only low dosages . Data are mean ± SD from three biological replicates . 1 - way ANOVA followed by Dunnett ' s multiple comparison tests : * P < 0 . 5 ; * * P < 0 . 01 ; * * * P < 0 .

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 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 1C Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ±SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin and #2 Tunicamycin.D, E Dose-response analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX used only low dosages. Data are mean ± SD from three biological replicates. 1-way ANOVA followed by Dunnett's multiple comparison tests: *P<0.5; **P<0.01; ***P<0.001


Figure 2B , C HEK293T cells were treated for 24 h and general RNA and protein synthesis were monitored by the incorporation of 5 - ethynyl uridine ( EU ) and O - propargyl - puromycin ( OPP ) , respectively . ActD ( 5 μM ) and CHX ( 350 μM ) were used for 3 h as positive control to block RNA and protein synthesis , respectively . Data are from three biological replicates and represented as mean ± SEM . ( B ) Two - tailed , unpaired t - test ; * * * P < 0 . 001 , * * P = 0 . 0047 . Concentration used : 1 μM GELD , 10 μM ERY , SPL and FSK . G Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated and treated for 24 h . Mutational analysis of near - cognate CUG start codon from three biological replicates and four technical replicates , n = 12 . Data are mean ± SEM . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * * * * P < 0 . 0001 . Concentration used : 1 μM GELD , 10 μM SPL and FSK .

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 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

Figure 2B, C HEK293T cells were treated for 24 h and general RNA and protein synthesis were monitored by the incorporation of 5-ethynyl uridine (EU) and O-propargyl-puromycin (OPP), respectively. ActD (5 μM) and CHX (350 μM) were used for 3 h as positive control to block RNA and protein synthesis, respectively. Data are from three biological replicates and represented as mean ± SEM. (B) Two-tailed, unpaired t-test; ***P<0.001, **P = 0.0047. Concentration used: 1 μM GELD, 10 μM ERY, SPL and FSK.G Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, ****P<0.0001. Concentration used: 1 μM GELD, 10 μM SPL and FSK.


Figure 3C Relative expression level of Sqstm1 / p62 , Lc3 , Tfeb , Hspb8 and Bag3 mRNAs in NSC34 cells treated for 24 h . Data are mean ± SEM from three biological replicates . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * * * P < 0 . 001 . Dotted lines represent RNA expression in the DMSO control . Concentration used : 1 μM GELD , 10 μM ERY , SPL , FSK . G Lysates from NSC34 cells transfected with scrambled - GPx100 , treated for 24 h with SPL and / or 10 mM 3MA and immunoblotted for scrambled - GP expression . Quantification from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test , * * * * P < 0 . 0001 for SPL vs DMSO and 3MA vs DMSO , * * * P = 0 . 0008 SPL + 3MA vs SPL . Concentration used : 1 μM GELD , 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 O O O O O O O O O O O O O O O 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

Figure 3C Relative expression level of Sqstm1/p62, Lc3, Tfeb, Hspb8 and Bag3 mRNAs in NSC34 cells treated for 24 h. Data are mean ± SEM from three biological replicates. 2-way ANOVA followed by Dunnett's multiple comparisons test, ***P<0.001. Dotted lines represent RNA expression in the DMSO control. Concentration used: 1 μM GELD, 10 μM ERY, SPL, FSK.G Lysates from NSC34 cells transfected with scrambled-GPx100, treated for 24 h with SPL and/or 10 mM 3MA and immunoblotted for scrambled-GP expression. Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001 for SPL vs DMSO and 3MA vs DMSO, ***P = 0.0008 SPL + 3MA vs SPL. Concentration used: 1 μM GELD, 10 μM SPL


Figure 4B Lysates from HEK293T cells , transfected with 2R or 66R and treated for 24 h , immunoblotted using antibody for poly - GA expression ( HA - tagged ) . Quantification from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test , * * * * P < 0 . 0001 , * * P = 0 . 0027 H89 vs DMSO , * * * P = 0 . 0004 FSK + H89 vs DMSO , * P = 0 . 0234 CLZ vs DMSO , * P = 0 . 0209 FSK + H89 vs FSK . Concentration used : 2 . 5 μM H89 , 10 μM FSK , CLZ . F Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated and treated with H89 for 24 h . Mutational analysis of near - cognate CUG start codon from three biological replicates and four technical replicates , n = 12 . Data are mean ± SEM . 2 - way ANOVA followed by Dunnett ' s multiple comparisons test , * P = 0 . 0130 WT H89 vs WT DMSO , * * * * P < 0 . 0001 , CUG mutated H89 vs CUG mutated DMSO . Concentration used : 2 . 5 μM H89 ,

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 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

Figure 4B Lysates from HEK293T cells, transfected with 2R or 66R and treated for 24 h, immunoblotted using antibody for poly-GA expression (HA-tagged). Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001, **P = 0.0027 H89 vs DMSO, ***P = 0.0004 FSK + H89 vs DMSO, *P = 0.0234 CLZ vs DMSO, *P = 0.0209 FSK + H89 vs FSK. Concentration used: 2.5 μM H89, 10 μM FSK, CLZ.F Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated with H89 for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, *P = 0.0130 WT H89 vs WT DMSO, ****P<0.0001, CUG mutated H89 vs CUG mutated DMSO. Concentration used: 2.5 μM H89,


Figure 5A , B Lysates from HEK293T cells , transfected with RNAi scrambled or RNAi PRKACA ( 72 h ) and 2R or 66R ( 24 h ) , immunoblotted for PKA - Cα and poly - GA ( HA - tag ) expression . α - Tubulin used as loading control . ( B ) Quantification of PKA - Cα and poly - GA expression from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test . PKA - Cα : ns siScr 66R vs siScr 2R , * * * P = 0 . 0002 siPRKACA 66R vs siScr 2R , * * * P = 0 . 0003 siPRKACA 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACA 66R vs siScr 2R , * * P = 0 . 0062 siPRKACA 66R vs siScr 66R . C , D Lysates from HEK293T cells , transfected with RNAi scrambled or RNAi PRKACB ( 72 h ) and 2R or 66R ( 24 h ) , immunoblotted PKA - Cβ and poly - GA ( HA tag ) expression . α - Tubulin used as loading control . ( D ) Quantification of PKA - Cβ and poly - GA expression from three biological replicates . Data are mean ± SEM . Two - tailed , unpaired t - test . PKA - Cβ : ns siScr 66R vs siScr 2R , * * P = 0 . 0049 siPRKACB 66R vs siScr 2R , * * P = 0 . 0091 siPRKACB 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACB 66R vs siScr 2R , * * * P = 0 . 0007 siPRKACB 66R vs siScr 66R . E

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

Figure 5A, B Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACA (72 h) and 2R or 66R (24 h), immunoblotted for PKA-Cα and poly-GA (HA-tag) expression. α-Tubulin used as loading control. (B) Quantification of PKA-Cα and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cα: ns siScr 66R vs siScr 2R, ***P = 0.0002 siPRKACA 66R vs siScr 2R, ***P = 0.0003 siPRKACA 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACA 66R vs siScr 2R, **P = 0.0062 siPRKACA 66R vs siScr 66R . C, D Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACB (72 h) and 2R or 66R (24 h), immunoblotted PKA-Cβ and poly-GA (HA tag) expression. α-Tubulin used as loading control. (D) Quantification of PKA-Cβ and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cβ: ns siScr 66R vs siScr 2R, **P = 0.0049 siPRKACB 66R vs siScr 2R, **P = 0.0091 siPRKACB 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACB 66R vs siScr 2R, ***P = 0.0007 siPRKACB 66R vs siScr 66R. E


Figure 6A , B Representative graphs showing the climbing ability flies carrying the UAS - ( G4C2 ) x36 construct in neurons using the Elav - gal4 promoter upon treatment with H89 10 µM diluted with 0 . 1 % of DMSO and 5 % sucrose in PBS . The wild - type ( w1118 ) line was used as control . ( A ) Female flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 6th day of treatment * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . ( B ) Male flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 6th day of treatment . * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . Experiments were performed three times , N = 15 adult flies were used for each genotype , sex and treatment ( N tot = 45 ) . C , D Representative graphs showing climbing activity of flies co - expressing the UAS - Pka - RNAi construct ( 1 , v330111 ) in combination with UAS - ( G4C2 ) x36 in neurons using the Elav - gal4 promoter . The wild - type ( w1118 ) line was used as control . ( C ) Female flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 9th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . ( D ) Male flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for the 7th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . Experiments were performed twice with different RNA interfering sequence , N = 15 adult flies were used for each genotype and sex ( N tot = 30 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Representative graphs showing the climbing ability flies carrying the UAS-(G4C2)x36 construct in neurons using the Elav-gal4 promoter upon treatment with H89 10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45). C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2)x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30).