( B ) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets . Yellow bar represents enrichment within NPC / GPC genes , green bar within neuronal genes , blue bar within astrocytic genes and red bar within oligodendrocytic genes . SOX10 ChIP - seq data from [

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(B) Bar graph shows enrichment of genes bound by SOX10 in rat oligodendrocytes within the differentially expressed gene sets. Yellow bar represents enrichment within NPC/GPC genes, green bar within neuronal genes, blue bar within astrocytic genes and red bar within oligodendrocytic genes. SOX10 ChIP-seq data from [37


( F ) Expression of Nfia , Ehf and Zbtb3 among spinal cord cells analyzed with scRNA - seq . Red color indicates cells with RPKM values above 55 for Nfia , 5 for Zbtb3 and 5 for Ehf . n = 3 for SOX9 and 2 for SOX3 . P - values ( phyper , R ) were calculated from the total number of genes for mm10 ( 23 ´ 389 )

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(F) Expression of Nfia, Ehf and Zbtb3 among spinal cord cells analyzed with scRNA-seq. Red color indicates cells with RPKM values above 55 for Nfia, 5 for Zbtb3 and 5 for Ehf. n=3 for SOX9 and 2 for SOX3. P-values (phyper, R) were calculated from the total number of genes for mm10 (23´389)


( A ) Luc - reporter assays in P19 cells . Luc - reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA ; an activation that could be efficiently blocked by SOX3 . No activation of these reporters could be detected with SOX10 or SOX11 . ( B ) Luc - reporters containing SOX9 and SOX10 bound DNA - regions around the astrocyte genes Aldh1l1 and Atp1a2 , which are not expressed in SOX10 + oligodendrocytes , could not be activated by SOX9 or SOX10

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(A) Luc-reporter assays in P19 cells. Luc-reporters containing SOX3 and SOX9 bound CRMs around the astrocyte specific genes Fgfbp3 and Ttyh1 were strongly activated by SOX9 in synergy with NFIA; an activation that could be efficiently blocked by SOX3. No activation of these reporters could be detected with SOX10 or SOX11. (B) Luc-reporters containing SOX9 and SOX10 bound DNA-regions around the astrocyte genes Aldh1l1 and Atp1a2, which are not expressed in SOX10+ oligodendrocytes, could not be activated by SOX9 or SOX10


( C ) Luc - reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10 . No activation could be detected with SOX11 . ( D ) Luc - reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2 , which are sequentially bound by SOX3 and SOX11 , could be activated by SOX11 and efficiently repressed by SOX3 . No activation could be detected with

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(C) Luc-reporters containing SOX9 and SOX10 bound CRMs around the oligodendrocyte specific genes Plekhb1 and Mid2 were activated in an additive manner by SOX9 and SOX10. No activation could be detected with SOX11. (D) Luc-reporters containing SOX3 and SOX11 bound CRMs of the neuronal genes Tubb3 and Lhx2, which are sequentially bound by SOX3 and SOX11, could be activated by SOX11 and efficiently repressed by SOX3. No activation could be detected with SOX10


( E ) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes . Scale bar , 25µm . ( F ) Statistical view of Fig . 5E . Bars represents mean values ( of 3 experiments ) of cells expressing GFAP or SOX10 in GFP or Sox3 - transduced cells . Error bars in figure 5 indicate standard deviation of 3 - 7 individual samples and three - star significance > 0 . 001 ( t - test ) . ( * ) 0 , 05 < P < 0 , 01 ; ( * * ) 0 , 01 < P < 0 , 001 ; ( * * * ) P < 0 ,

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(E) Forced expression of SOX3 for 96 hrs in differentiating GPCs blocked the formation of astrocytes but not oligodendrocytes. Scale bar, 25µm. (F) Statistical view of Fig. 5E. Bars represents mean values (of 3 experiments) of cells expressing GFAP or SOX10 in GFP or Sox3-transduced cells. Error bars in figure 5 indicate standard deviation of 3-7 individual samples and three-star significance > 0.001 (t-test). (*) 0,05<P<0,01; (**) 0,01<P<0,001; (***) P<0,001


( A ) SOX3 over - expression experiments in chicken spinal cord at E4 . 48 hrs post electroporation , transfected side showed decreased expression of FGFR2 , NFIA and Fgfr3 . Bar : 50µm . ( B ) 24 hrs after miss - expression of a dominant negative version of SOXB1 ( dnSOXB1 ) , premature expression of FGFR2 , NFIA and Fgfr3 were found in the transfected side . Bar : 40µm . ( C ) 24 hrs after SOX9 over - expression in chicken spinal cord ( at E4 ) , increased expression of FGFR2 , NFIA and Fgfr3 was shown compared to the un - transfected control side . Bar : 40µm . ( D ) Sox3 and Sox9 co - electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2 , NFIA and Fgfr3 levels were comparable to the un - transfected control side . Embryos were analyzed 24 hrs post electroporation . Bar : 40µm . ( E

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(A) SOX3 over-expression experiments in chicken spinal cord at E4. 48 hrs post electroporation, transfected side showed decreased expression of FGFR2, NFIA and Fgfr3. Bar: 50µm. (B) 24 hrs after miss-expression of a dominant negative version of SOXB1 (dnSOXB1), premature expression of FGFR2, NFIA and Fgfr3 were found in the transfected side. Bar: 40µm. (C) 24 hrs after SOX9 over-expression in chicken spinal cord (at E4), increased expression of FGFR2, NFIA and Fgfr3 was shown compared to the un-transfected control side. Bar: 40µm. (D) Sox3 and Sox9 co-electroporation in chicken spinal cord at E4 could rescue the effect of SOX9 overexpression and FGFR2, NFIA and Fgfr3 levels were comparable to the un-transfected control side. Embryos were analyzed 24 hrs post electroporation. Bar: 40µm. (E


( B ) Representative images of cells released from mitosis for 1 , 2 or 3h in EdU - containing medium . Top , DNA staining with DAPI ; Bottom , EdU immunofluorescence . L , M and H indicate cells with low , medium or high levels of EdU incorporation . The barplot shows the quantification of the proportion of replicating cells at each time point .

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(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.


Y - axis , z - score ; X - axis , timepoints . A indicates asynchronous cells , M mitotic cells and the numbers represent the minutes after release from the mitotic block ) . Dots show individual replicates and lines averages ( n = 3 for A and M ; n = 2 for each post - mitotic sample ) . Pre - mRNA levels are shown for pluripotency genes ( Oct4 , Klf4 , Esrrb ) and cell cycle regulators ( E2f1 , Cdk2 , Rb ) ; mRNA levels for individual histone genes ( only averages are shown ) .

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Y-axis, z-score; X-axis, timepoints. A indicates asynchronous cells, M mitotic cells and the numbers represent the minutes after release from the mitotic block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone genes (only averages are shown).


( C ) Percentage of genes presenting at least 1 TF binding site as detected by ChIP - seq within 25kb centred on the TSS , calculated for each gene category shown in ( A ) .

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(C) Percentage of genes presenting at least 1 TF binding site as detected by ChIP-seq within 25kb centred on the TSS, calculated for each gene category shown in (A).


( B ) Heatmaps presented for untreated ( - IAA ; left ) or CTCF - depleted ( + IAA ; right ) CTCF - AID cells .

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(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.


( A ) Gene expression changes ( z - score ) in asynchronous cells and during post - mitotic release of CTCF - AID cells treated or not with IAA to deplete CTCF ( blue , presence of CTCF ; red , absence ) . Samples are indicated on the X - axis ( A : asynchronous cells ; M : mitotic cells ; numbers : minutes since release from mitotic block ) . Six gene groups are shown , based on the time of their response to CTCF depletion ( asy , late , early ) and the direction of the transcriptional change ( up , down ) . The number of genes of each group is indicated . The yellow shadow areas indicate times showing expression changes . The boxplots show the median ( bar ) , 25 - 75 % percentiles ( box ) and 1 . 5 - fold inter - quartile range ( whiskers ) .

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(A) Gene expression changes (z-score) in asynchronous cells and during post-mitotic release of CTCF-AID cells treated or not with IAA to deplete CTCF (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous cells; M: mitotic cells; numbers: minutes since release from mitotic block). Six gene groups are shown, based on the time of their response to CTCF depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times showing expression changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).


( C ) Average binding profile ( X - axis , reads per million ) of CTCF ( top ) and the Cohesin subunit SMC1 ( bottom ) , across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells ( left ) or either late or early upon mitotic release ( middle and right , respectively ) . Interphase is shown in blue and mitosis in red .

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(C) Average binding profile (X-axis, reads per million) of CTCF (top) and the Cohesin subunit SMC1 (bottom), across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells (left) or either late or early upon mitotic release (middle and right, respectively). Interphase is shown in blue and mitosis in red.


B . Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C - terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control .

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B. Western blot of PTEN variants in a panel of human cancer cell lines using a rabbit monoclonal antibody against the C-terminal region of PTEN The PTEN null prostate cancer cell line PC3 was used as a negative control.


D . Verification of expression of FLAG - tagged PTENα , PTENβ , and PTENε in PtenFLAG knock - in mice . Various tissues from PtenFLAG knock - in mice or control wild - type mice were lysed for immunoprecipitation with anti - FLAG M2 magnetic beads before being immunoblotted with the anti - PTENα

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D. Verification of expression of FLAG-tagged PTENα, PTENβ, and PTENε in PtenFLAG knock-in mice. Various tissues from PtenFLAG knock-in mice or control wild-type mice were lysed for immunoprecipitation with anti-FLAG M2 magnetic beads before being immunoblotted with the anti-PTENα antibody


B . Reduction of PTENε in response to eIF2A knockdown . Hela cells were infected with lentivirus expressing eIF2A shRNA , eIF5 shRNA , or scramble shRNA separately . The knockdown of eIF2A or eIF5 was validated by western blot ( left panel ) . Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN , and GAPDH ( right panel ) .

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B. Reduction of PTENε in response to eIF2A knockdown. Hela cells were infected with lentivirus expressing eIF2A shRNA, eIF5 shRNA, or scramble shRNA separately. The knockdown of eIF2A or eIF5 was validated by western blot (left panel). Lysates of Hela cells were collected and subjected to western blot analysis with antibodies against PTEN, and GAPDH (right panel).


C . Overexpression of eIF2A induces the relative expression of PTENε . FLAG - tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN . GAPDH was used as a loading control .

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C. Overexpression of eIF2A induces the relative expression of PTENε. FLAG-tagged eIF2A or eIF5 were overexpressed in DU145 cells before western blot analysis of PTENε expression with an antibody against PTEN. GAPDH was used as a loading control.


C . The transfected Hela PTEN - / - cells were stained with an anti - β - catenin antibody and DAPI before being imaged with confocal microscopy .

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C. The transfected Hela PTEN-/- cells were stained with an anti-β-catenin antibody and DAPI before being imaged with confocal microscopy.


To determine the sequences that are critical for the membrane localization of PTENε . Hela were transfected with constructs Thirty - six hours after transfection , transfected Hela cells were stained with anti - β - catenin antibody and DAPI , followed by imaging with confocal microscopy . The scale bars represent 5 μm ( F ) .

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To determine the sequences that are critical for the membrane localization of PTENε. Hela were transfected with constructs Thirty-six hours after transfection, transfected Hela cells were stained with anti-β-catenin antibody and DAPI, followed by imaging with confocal microscopy. The scale bars represent 5 μm (F).


The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP , E - cadherin , and β - tubulin antibodies ( G ) . C : cytoplasm . M : cell plasma membrane .

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The transfected HEK293 cells were conducted with cell fractionation before western blotting with GFP, E-cadherin, and β-tubulin antibodies (G). C: cytoplasm. M: cell plasma membrane.


D . Images of immunofluorescence staining for PTENε and its interacting proteins ( VASP and FSCN1 ) . Hela PTEN - / - cells were co - transfected by a plasmid expressing C - terminal GFP - tagged PTENε with FLAG - tagged targets ( FSCN1 , VASP ) separately , followed by staining with an anti - FLAG antibody and DAPI , and were imaged by confocal microscopy .

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D. Images of immunofluorescence staining for PTENε and its interacting proteins (VASP and FSCN1). Hela PTEN-/- cells were co-transfected by a plasmid expressing C-terminal GFP-tagged PTENε with FLAG-tagged targets (FSCN1, VASP) separately, followed by staining with an anti-FLAG antibody and DAPI, and were imaged by confocal microscopy.


E . The cell plasma membrane localization of PTENε is required for its interaction with downstream targets . FLAG - tagged CDC42 , VASP , ACTR2 , or FSCN1 plasmid was co - transfected with a plasmid expressing PTENε with or without 69 - 81aa deletion carrying C - terminal S - tag and HA tag in HEK293 cells . Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag .

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E. The cell plasma membrane localization of PTENε is required for its interaction with downstream targets. FLAG-tagged CDC42, VASP, ACTR2, or FSCN1 plasmid was co-transfected with a plasmid expressing PTENε with or without 69-81aa deletion carrying C-terminal S-tag and HA tag in HEK293 cells. Cell lysates were incubated with S protein agarose before western blotting with antibodies against the FLAG tag and HA tag.


( H , I ) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His - PTENε or without purified PTENε protein as a control , followed by immunoblotting with antibodies against p - ACTR2 ( Thr237 and Thr238 ) and p - VASP ( Ser157 ) . Purified proteins ( PTENε , PKA , NIK , ACTR , and VASP ) were detected by anti - His antibody ( ZSGB - BIO , TA - 02 ) .

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(H, I) Purified ACTR2 or VASP was phosphorylated in vitro by NIK or PKA and subsequently used in a phosphatase assay with His-PTENε or without purified PTENε protein as a control, followed by immunoblotting with antibodies against p-ACTR2 (Thr237 and Thr238) and p-VASP (Ser157). Purified proteins (PTENε, PKA, NIK, ACTR, and VASP) were detected by anti-His antibody (ZSGB-BIO, TA-02).


A . Immunofluorescence staining of F - actin in PTENε or PTENε Y210L overexpressed Hela PTEN - / - cells and control cells . PTENε - GFP , PTENε Y210L - GFP , or mock construct was introduced into HeLa PTEN - / - cells respectively . The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy . The scale bars represent 5 μm ( left panel ) . White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane . B . Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t - test . * * * * , p < 0 . 001 .

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A. Immunofluorescence staining of F-actin in PTENε or PTENε Y210L overexpressed Hela PTEN-/- cells and control cells. PTENε-GFP, PTENε Y210L-GFP, or mock construct was introduced into HeLa PTEN-/- cells respectively. The transfected cells were stained with Phalloidin and DAPI before being imaged with confocal microscopy. The scale bars represent 5 μm (left panel). White arrows shown in the magnified immunofluorescence images indicate the filopodia in the cell membrane. B. Quantification of the number of filopodia per cell by FiloQuant software and the data are presented as mean ± SD of three independent experiments and were analyzed with the unpaired t-test. ****, p<0.001.


G . Left panel : immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells . Middle panel : representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model ( n = 4 ) . H & E , hematoxylin - eosin staining . The scale bars represent 600 mm ( upper panel ) and 500μm ( lower panel ) separately . Right panel : the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules ( data are presented as mean ± SD based on three independent replicates ) . The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group . * * * * , p < 0 . 0001 in the unpaired t - test .

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G. Left panel: immunoblotting analyses were used to verify the level of overexpressed PTENε or PTENε Y210L in B16 cells. Middle panel: representative images showing the lung metastasis of PTENε or PTENε Y210L overexpressed B16 cells or control cells in the mouse pulmonary metastasis model (n=4). H & E, hematoxylin-eosin staining. The scale bars represent 600 mm (upper panel) and 500μm (lower panel) separately. Right panel: the representative bar graphs showing the quantification of tumor metastatic ability was displayed by measuring the number of tumor nodules (data are presented as mean ± SD based on three independent replicates). The overexpression of PTENε attenuated tumor metastatic ability compared with the vector control group. ****, p<0.0001 in the unpaired t-test.


( A and B ) THP1 - derived macrophages were treated with the TLR ligands Pam3 , lipopolysaccharide ( LPS ) , or FSL - 1 and analyzed by RNA - seq 8 h later . Circos plots show genome - wide differential expression of protein - coding genes ( A ) and non - protein - coding genes ( B ) between untreated and treated macrophages . The outermost to innermost circles show downregulated ( blue ) or upregulated ( red ) genes in cells stimulated with FSL - 1 , LPS , and Pam3 , respectively . Chromosomes are indicated by the numbers 1 - 22 and X and Y .

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(A and B) THP1-derived macrophages were treated with the TLR ligands Pam3, lipopolysaccharide (LPS), or FSL-1 and analyzed by RNA-seq 8 h later. Circos plots show genome-wide differential expression of protein-coding genes (A) and non-protein-coding genes (B) between untreated and treated macrophages. The outermost to innermost circles show downregulated (blue) or upregulated (red) genes in cells stimulated with FSL-1, LPS, and Pam3, respectively. Chromosomes are indicated by the numbers 1-22 and X and Y.


( F ) RT - qPCR validation of differentially expressed lncRNAs and mRNAs in THP1 - derived macrophages stimulated for 8 h with flagellin ( FLA ) , FSL - 1 , imiquimod , LPS , Pam3 , or poly ( I : C ) . Heat map shows the fold change in expression of 13 mRNAs ( left ) and lncRNAs ( right ) .

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(F) RT-qPCR validation of differentially expressed lncRNAs and mRNAs in THP1-derived macrophages stimulated for 8 h with flagellin (FLA), FSL-1, imiquimod, LPS, Pam3, or poly(I:C). Heat map shows the fold change in expression of 13 mRNAs (left) and lncRNAs (right).


( A ) Activation and suppression of nearby genes by eight cis - acting lncRNAs . RT - qPCR analysis of THP1 - derived macrophages transduced with lentiviruses expressing non - targeting control ( NC ) shRNA or shRNAs targeted to the indicated lncRNAs and protein - coding genes . Mean ± SD of n = 3 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by Student ' s t test .

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(A) Activation and suppression of nearby genes by eight cis-acting lncRNAs. RT-qPCR analysis of THP1-derived macrophages transduced with lentiviruses expressing non-targeting control (NC) shRNA or shRNAs targeted to the indicated lncRNAs and protein-coding genes. Mean ± SD of n = 3. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001 by Student's t test.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 6 IL - 6 ELISA quantification ( C ) of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . ELISA were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-6 IL-6 ELISA quantification (C) of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. ELISA were performed at 10 h post-infection.


Silencing of cis - regulated gene pairs affects GBS - induced expression of IL - 1α ( D ) . RT - qPCR analysis of THP1 - derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h , infected with GBS for 1 h , and then treated with gentamicin for 30 min to terminate infection . RT - qPCR were performed at 10 h post - infection .

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Silencing of cis-regulated gene pairs affects GBS-induced expression of IL-1α (D). RT-qPCR analysis of THP1-derived macrophages transduced with the indicated NC or targeting shRNAs for 72 h, infected with GBS for 1 h, and then treated with gentamicin for 30 min to terminate infection. RT-qPCR were performed at 10 h post-infection.


lnc - MARCKS ( B ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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lnc-MARCKS (B) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


, MARCKS ( C ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

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, MARCKS (C) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


IL - 6 ( D ) expressions were quantified in cells stably expressing dCas9 / KRAB / BFP and the NC or sgRNAs .

IL-6 (D) expressions were quantified in cells stably expressing dCas9/KRAB/BFP and the NC or sgRNAs.


Overexpression of lnc - MARCKS decreases MARCKS expression RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . Lnc - MARCKS and MARCKS expression ( F )

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Overexpression of lnc-MARCKS decreases MARCKS expression RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. Lnc-MARCKS and MARCKS expression (F)


Overexpression of lnc - MARCKS enhances IL - 6 expression . RT - qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector ( E . V . ) or lnc - MARCKS , treated with PMA overnight , and left unstimulated or stimulated with Pam3 for 8 h . IL - 6 expression ( G ) .

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Overexpression of lnc-MARCKS enhances IL-6 expression. RT-qPCR analysis of THP1 cells transduced with lentiviruses expressing empty vector (E.V.) or lnc-MARCKS, treated with PMA overnight, and left unstimulated or stimulated with Pam3 for 8 h. IL-6 expression (G).


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect lnc - MARCKS ( H )

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect lnc-MARCKS (H)


lnc - MARCKS regulates MARCKS in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS ( I ) ,

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lnc-MARCKS regulates MARCKS in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect MARCKS (I),


lnc - MARCKS regulates IL - 6 in primary macrophages . Primary macrophages were transfected with NC or lnc - MARCKS antisense oligo to knock down lnc - MARCKS expression . 48 h later , cells were stimulated with Pam3 for 8 h and harvested to detect IL - 6 expressions ( J ) .

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lnc-MARCKS regulates IL-6 in primary macrophages. Primary macrophages were transfected with NC or lnc-MARCKS antisense oligo to knock down lnc-MARCKS expression. 48 h later, cells were stimulated with Pam3 for 8 h and harvested to detect IL-6 expressions (J).


( A ) ROCKI is recruited to the MARCKS promoter in Pam3 - stimulated THP1 - derived macrophages . ChIRP analysis of THP1 - derived macrophages treated with Pam3 for 8 h . ChIRP assays were performed using a non - specific lacZ probe or probes specific for lnc - MARCKS . Specificity of ROCKI probes ( left ) , and enrichment at the MARCKS promoter ( right ) .

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(A) ROCKI is recruited to the MARCKS promoter in Pam3-stimulated THP1-derived macrophages. ChIRP analysis of THP1-derived macrophages treated with Pam3 for 8 h. ChIRP assays were performed using a non-specific lacZ probe or probes specific for lnc-MARCKS. Specificity of ROCKI probes (left), and enrichment at the MARCKS promoter (right).


( B ) Mass spectrometry identifies APEX1 as a candidate ROCKI - binding protein in THP1 - derived macrophages . APEX1 MS / MS count represents the ratio between the number of APEX1 peptides and all peptides detected . Mean of n = 2 samples . ROCKI and ROCKI - AS represent ROCKI and antisense strand of ROCKI transcripts , respectively .

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(B) Mass spectrometry identifies APEX1 as a candidate ROCKI-binding protein in THP1-derived macrophages. APEX1 MS/MS count represents the ratio between the number of APEX1 peptides and all peptides detected. Mean of n = 2 samples. ROCKI and ROCKI-AS represent ROCKI and antisense strand of ROCKI transcripts, respectively.


( C ) ROCKI inhibition of MARCKS expression is dependent on APEX1 . RT - qPCR analysis of MARCKS , ROCKI , and APEX1 expression in THP1 - derived macrophages overexpressing ROCKI and / or depleted of APEX1 .

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(C) ROCKI inhibition of MARCKS expression is dependent on APEX1. RT-qPCR analysis of MARCKS, ROCKI, and APEX1 expression in THP1-derived macrophages overexpressing ROCKI and/or depleted of APEX1.


( D ) APEX1 specially binds to ROCKI . Western blot analysis of APEX1 in lacZ , or ROCKI pull - down fractions . Vimentin , which non - specifically binds to all RNA probes , is shown as a loading control .

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(D) APEX1 specially binds to ROCKI. Western blot analysis of APEX1 in lacZ, or ROCKI pull-down fractions. Vimentin, which non-specifically binds to all RNA probes, is shown as a loading control.


( E ) ROCKI associates with APEX1 . Left : RT - qPCR analysis of ROCKI present in anti - APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3 - treated THP1 - derived macrophages . Right : APEX1 western blot of the same immunoprecipitates .

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(E) ROCKI associates with APEX1. Left: RT-qPCR analysis of ROCKI present in anti-APEX1 antibody or control IgG immunoprecipitates from nuclear lysates of Pam3-treated THP1-derived macrophages. Right: APEX1 western blot of the same immunoprecipitates.


ChIP analysis of THP1 - derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter . RT - qPCR analysis of fragments of the MARCKS promoter region ( a and b ) or a non - specific region ( nc ) co - immunoprecipitated with anti - APEX1 antibody .

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ChIP analysis of THP1-derived macrophages demonstrates APEX1 recruitment to the MARCKS promoter. RT-qPCR analysis of fragments of the MARCKS promoter region (a and b) or a non-specific region (nc) co-immunoprecipitated with anti-APEX1 antibody.


ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA ( NC ) or ROCKI shRNA and treated with Pam3 for 8 h . The y - axis shows the percentage of enrichment in normalized to input . An unrelated region of the ACTB2 promoter was amplified as a control .

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ChIP analysis of APEX1 recruitment to the MARCKS promoter was performed except the cells expressed control shRNA (NC) or ROCKI shRNA and treated with Pam3 for 8 h. The y-axis shows the percentage of enrichment in normalized to input. An unrelated region of the ACTB2 promoter was amplified as a control.


Left : Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti - APEX1 antibody immunoprecipitates from NC ( control ) or ROCKI knockdown THP1 - derived macrophages . Right : RT - qPCR analysis of ROCKI in the same immunoprecipitates .

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Left: Western blot analysis of APEX1 and HDAC1 in control rabbit IgG or anti-APEX1 antibody immunoprecipitates from NC (control) or ROCKI knockdown THP1-derived macrophages. Right: RT-qPCR analysis of ROCKI in the same immunoprecipitates.


Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter . ChIP - qPCR analysis of HDAC1 was performed using an anti - HDAC1 antibody .

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Knockdown of ROCKI decreased HDAC1 enrichment on the MARCKS promoter. ChIP-qPCR analysis of HDAC1 was performed using an anti- HDAC1 antibody.


Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter . ChIP - qPCR analysis of H3K27ac was performed using an anti - H3K27ac antibody .

Knockdown of ROCKI enhances H3K27ac deposition on the MARCKS promoter. ChIP-qPCR analysis of H3K27ac was performed using an anti-H3K27ac antibody.


Heatmap of representative genes commonly upregulated in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages .

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Heatmap of representative genes commonly upregulated in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages.


RT - qPCR validation of inflammatory gene expression in ROCKI - overexpressing and MARCKS - knockdown THP1 - derived macrophages stimulated with Pam3 for 8 h . E . V . , empty vector ; NC , control shRNA .

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RT-qPCR validation of inflammatory gene expression in ROCKI-overexpressing and MARCKS-knockdown THP1-derived macrophages stimulated with Pam3 for 8 h. E.V., empty vector; NC, control shRNA.


MARCKS is a negative regulator of Ca2 + signaling . Fluo - 4 fluorescence of MARCKS , ROCKI , or NC ( control ) shRNA - expressing THP1 - derived macrophages before and after treatment with PMA and Pam3 . Triton X - 100 was added to obtain the maximal [ Ca2 + ] signal . Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence ( ΔF / Fmax ) .

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MARCKS is a negative regulator of Ca2+ signaling. Fluo-4 fluorescence of MARCKS, ROCKI, or NC (control) shRNA-expressing THP1-derived macrophages before and after treatment with PMA and Pam3. Triton X-100 was added to obtain the maximal [Ca2+] signal. Fluorescence levels are expressed as the change in signal normalized to the maximal fluorescence (ΔF/Fmax).


( A ) QTL signals for ROCKI ( LINC01268 ) gene expression in whole blood ( top , black ) , H3K27ac promoter signal in monocytes ( middle , green ) , and H3K4me1 signal in monocytes ( bottom , orange ) in a 400 kb window around the MARCKS / ROCKI locus . The signals were strongly co - localized ( Pshared = . 97 , Pshared = . 98 ) suggesting they represent the same signal .

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(A) QTL signals for ROCKI (LINC01268) gene expression in whole blood (top, black), H3K27ac promoter signal in monocytes (middle, green), and H3K4me1 signal in monocytes (bottom, orange) in a 400 kb window around the MARCKS/ROCKI locus. The signals were strongly co-localized (Pshared=.97, Pshared=.98) suggesting they represent the same signal.


( C ) RT - qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M . tuberculosis - infected donors .

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(C) RT-qPCR analysis of ROCKI expression in dendritic cell samples from uninfected and M. tuberculosis-infected donors.


D , Affinity capture of proteins by OC43p and OC43 - SCR bound magnetic beads . Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies . OC43p - SCR - coated and uncoated beads ( Beads ) were used to control non - specific binding . Spinal cords were isolated from male ( M ) and female ( F ) rats ( n = 2 / group ) . Input protein lysates ( right side , in duplicates ) served as loading controls . Approximate molecular weights ( in kDa ) are indicated by arrows .

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D, Affinity capture of proteins by OC43p and OC43-SCR bound magnetic beads. Specific reactivity to Atp1a1 and Atp1a2 was detected by protein immunoblotting using specific primary antibodies. OC43p-SCR-coated and uncoated beads (Beads) were used to control non-specific binding. Spinal cords were isolated from male (M) and female (F) rats (n=2/group). Input protein lysates (right side, in duplicates) served as loading controls. Approximate molecular weights (in kDa) are indicated by arrows.


Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti - TAPBPL or isotype Ab .

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Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab.


Splenocytes from C57BL / 6 mice were freshly harvested . Resting and activated T cells , monocytes , macrophages , DCs and B cells were obtained as in Figure 2 . The resting and activated immune cells were stained with biotinylated TAPBPL - Ig or control Ig ， followed by streptavidin PE , as well as anti - CD4 , CD8 , CD11b , F4 / 80 , CD11c , B220 or CD19 antibody to identify immune cells . statistical analysis showing the binding TAPBPL - Ig or control Ig to freshly harvest and activated immune cells ( n = 3 ) . Significance was calculated by two ‐ way ANOVA with Tukey test . * P < 0 . 05 compared with control Ig . * * P < 0 . 05 compared with resting cells .

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Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells.


Splenic cells from C57BL / 6 mice were cultured with anti - CD3 antibody and anti - CD28 ( 0 . 5 µg / ml ) antibodies in the presence of graded doses of hTAPBPL - Ig or equimolar amounts of control Ig ( 3 . 75 and 5 . 63 µg / ml ) for 1 day The cells were analyzed for CD69 + , cells in CD4 and CD8 T cells . Representative flow cytometric and statistical analyses of the percentages of CD69 + , and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

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Splenic cells from C57BL/6 mice were cultured with anti-CD3 antibody and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 1 day The cells were analyzed for CD69+, cells in CD4 and CD8 T cells. Representative flow cytometric and statistical analyses of the percentages of CD69+, and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Splenic cells were labelled with CFSE and cultured in 96 - well plates that were precoated with anti - CD3 antibody and hTAPBPL - Ig or control Ig for 3 days as in ( A ) . The cells were analyzed for CFSE levels by CD4 + and CD8 + T cells . statistical analysis of ( D ) CD4 and ( E ) CD8 T cell proliferation .

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Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. statistical analysis of (D) CD4 and (E) CD8 T cell proliferation.


Purified human T cells were cultured with plate - bound anti - human CD3 antibody ( 1 µg / ml ) in the presence of graded doses of hTAPBPL - Ig ( 1 . 5 and 3 µg / ml ) or control Ig protein ( 1 . 5 and 3 µg / ml ) for 3 days . Cell proliferation was measured by [ 3H ] thymidine incorporation . The data are expressed as mean + SD ( n = 3 ) . Significance was calculated by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 compared with control Ig .

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Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.


Anti - hTAPBPL mAb ( clone 54 ) neutralized the inhibitory activity of hTAPBPL - Ig on T cell proliferation Significance in was calculated by one ‐ way ANOVA with Dunnett test and others by two ‐ tailed Student ' s t ‐ test . * P < 0 . 05 , anti - hTAPBPL Ab group was compared with isotype Ab group .

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Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on T cell proliferation Significance in was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.


DBA / 2J mice were injected s . c . with 1 × 105 P388 murine leukemia cells , followed by injection of the anti - hTAPBPL mAb ( 25 , 50 , or 100µg ) or isotype Ab ( 25 , 50 , or 100 µg ) 3 times per week . Tumors size was measured over time . The mean tumor diameter ( mm3 ) + S . D . at the indicated time points are shown .

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DBA/2J mice were injected s.c. with 1× 105 P388 murine leukemia cells, followed by injection of the anti-hTAPBPL mAb (25, 50, or 100µg) or isotype Ab (25, 50, or 100 µg) 3 times per week. Tumors size was measured over time. The mean tumor diameter (mm3) + S.D. at the indicated time points are shown.


Tumor infiltrating CD4 + and CD8 + T cells were examined by immunofluorescence .

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Tumor infiltrating CD4+ and CD8+ T cells were examined by immunofluorescence.


LDH assay was performed after 24 h of cold stress on ice . Data are presented as mean ± SEM ; n = 3 , biological replicates . * * * * p < 0 . 0001 , * * * p < 0 . 001 by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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LDH assay was performed after 24 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****p < 0.0001, ***p < 0.001 by one-way ANOVA followed by Dunnett's multiple comparison test.


Immunoblots of MICU1 and its mutants rescued by the lentivirus . Arrows indicate MICU1 or the dimer . * indicates the potential DTT - resistant dimer .

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Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer.


MMP was monitored by TMRE fluorescence at every 1 h . FCCP ( 200 µM ) and antimycin A ( 50 µM ) were used as pretreatments for 30 min . Data were normalized by basal state of WT A549 cells ( B - D ) Data at 1 h ( C ) , 5 h ( D are presented as mean ± SEM ; n = 4 ( B - D ) biological replicates , * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by paired T - test ( C ) , or one - way ANOVA followed by Dunnett ' s multiple comparison test (

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MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (D


LDH release was measured after cold stress for 24 h . FCCP ( 200 µM ) , antimycin A ( 50 µM ) , mitoQ and decylQ ( 500 nM ) were used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 4 ( G ) , biological replicates , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , by one - way ANOVA followed by Dunnett ' s or Bonferroni ' s multiple comparison test . whose raw value were normalized by the C11 - BODIPY 581 / 591 without cold stress of non - treatment , are presented as mean ± SEM ; n = 5 , biological replicates . * * p < 0 . 01 , * p < 0 . 05 , by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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LDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.


LDH release was measured after cold stress for 24 h . BAPTA - AM ( 10 µM ) was used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 3 , biological replicates , * * * * p < 0 . 0001 , by unpaired T - test .

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LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.


LDH release was measured after cold stress for 16 h . Data are presented as mean ± SEM ; n = 5 , biological replicates , * p < 0 . 05 , by unpaired T - test .

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LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 5, biological replicates, *p < 0.05, by unpaired T-test.


B : Silver nitrate staining of proteins pulled - down using the control peptide ( left ) or the FFAT peptide ( right ) , after SDS - PAGE . The two major differential bands are highlighted by

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B: Silver nitrate staining of proteins pulled-down using the control peptide (left) or the FFAT peptide (right), after SDS-PAGE. The two major differential bands are highlighted by arrows


C : Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction . Score : protein score based on the sum of the ion scores of all peptides identified ; Cov . ; percentage of the protein sequence covered by identified peptides ; Pep . : number of unique peptide sequences identified

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C: Tandem mass spectrometry result table showing the three top scored proteins identified in the FFAT peptide bound fraction. Score: protein score based on the sum of the ion scores of all peptides identified; Cov.; percentage of the protein sequence covered by identified peptides; Pep.: number of unique peptide sequences identified


E : Western blot analysis of proteins pulled - down using the control ( Ctrl ) peptide ( left ) or the FFAT ( FFAT ) peptide ( right ) . The Input , FT ( flow - through ) , and Elution fractions correspond to HeLa cell total protein extract , unbound proteins and bound proteins , respectively . Representative illustration of at least two independent experiments

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E: Western blot analysis of proteins pulled-down using the control (Ctrl) peptide (left) or the FFAT (FFAT) peptide (right). The Input, FT (flow-through), and Elution fractions correspond to HeLa cell total protein extract, unbound proteins and bound proteins, respectively. Representative illustration of at least two independent experiments


A : List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif ( see Appendix Table S1 for the full list ) . Proteins co - precipitated with GFP - MOSPD2 and GFP - MOSPD2 RD / LD proteins were identified by mass spectrometry ; proteins were filtered for their ability to bind MOSPD2 and not the RD / LD mutant . The sub - cellular localization of the proteins is indicated [ 53 , 65 - 69 ]

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A: List of the 5 top scored proteins interacting with MOSPD2 and known to contain a FFAT motif (see Appendix Table S1 for the full list). Proteins co-precipitated with GFP-MOSPD2 and GFP-MOSPD2 RD/LD proteins were identified by mass spectrometry; proteins were filtered for their ability to bind MOSPD2 and not the RD/LD mutant. The sub-cellular localization of the proteins is indicated [53,65-69]


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagged STARD3 ( B ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagged STARD3 (B) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD3NL ( C ) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed usin anti - ORP1 ( C ) , and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD3NL (C) Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed usin anti-ORP1 (C), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge ORP1L ( D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E ) , anti - ORP1 ( D ) ) , anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge ORP1L (D Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E), anti-ORP1 (D) ), anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) and Flag - tagge STARD11 ( E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti - Flag ( B , C - E anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies Immunoprecipitated material was analyzed using anti - Flag ( B , D - F and anti - GFP ( B - F )

B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) and Flag-tagge STARD11 (E Approximatively 5 µg of total protein extracts were analyzed by Western blot using anti-Flag (B, C-E anti-GFP (B-F), and anti-actin (B-F) antibodies Immunoprecipitated material was analyzed using anti-Flag (B, D-F and anti-GFP (B-F) antibodies


B - F : Immunoprecipitation ( GFP - Trap ) experiments between GFP - MOSPD2 ( WT and RD / LD mutant ) an HA - tagged PTPIP51 ( F ) ( WT and FFAT - deficient ) . Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti - HA ( F ) anti - GFP ( B - F ) , and anti - actin ( B - F ) antibodies . Immunoprecipitated material was analyzed using anti - Flag ( B , D - F ) , and anti - GFP ( B - F )

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B-F: Immunoprecipitation (GFP-Trap) experiments between GFP-MOSPD2 (WT and RD/LD mutant) an HA-tagged PTPIP51 (F) (WT and FFAT-deficient). Approximatively 5 µg of total protein extracts were analyzed by Western blot usin anti-HA (F) anti-GFP (B-F), and anti-actin (B-F) antibodies. Immunoprecipitated material was analyzed using anti-Flag (B, D-F ), and anti-GFP (B-F) antibodies


G : Immunoprecipitation ( anti - Flag ) experiment between Flag - tagged STARD3NL ( WT and FFAT - deficient ) and endogenous MOSPD2 . Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti - MOSPD2 , anti - STARD3NL and anti - actin antibodies

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G: Immunoprecipitation (anti-Flag) experiment between Flag-tagged STARD3NL (WT and FFAT-deficient) and endogenous MOSPD2. Proteins extracts and immunoprecipitated material were analyzed by Western blot using anti-MOSPD2, anti-STARD3NL and anti-actin antibodies


A : GFP - tagged MOSPD2 ( green ) expressing cells were labelled with an anti - MOSPD2 antibody ( magenta ) . The superposition of the green and magenta signals indicates that the anti - MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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A: GFP-tagged MOSPD2 (green) expressing cells were labelled with an anti-MOSPD2 antibody (magenta). The superposition of the green and magenta signals indicates that the anti-MOSPD2 antibody recognized efficiently MOSPD2 by immunofluorescence Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


B : Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells ( WT ) and in HeLa cells expressing a control shRNA ( shCtrl ) and two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Quantification of MOSPD2 protein level is shown below . Means and error bars ( SD ) are shown . n : three independent experiments

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B: Western blot analysis of MOSPD2 and VAP proteins in control HeLa cells (WT) and in HeLa cells expressing a control shRNA (shCtrl) and two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Quantification of MOSPD2 protein level is shown below. Means and error bars (SD) are shown. n: three independent experiments


C : Endogenous MOSPD2 ( green ) was labelled in control HeLa cells ( WT ) , in cells expressing a control shRNA ( shCtrl ) , and in cells expressing two individual shRNAs targeting MOSPD2 ( shMOSPD2 - α or shMOSPD2 - β ) . Scale bar : 10 µm

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C: Endogenous MOSPD2 (green) was labelled in control HeLa cells (WT), in cells expressing a control shRNA (shCtrl), and in cells expressing two individual shRNAs targeting MOSPD2 (shMOSPD2-α or shMOSPD2-β). Scale bar: 10 µm


D : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing the ER marker GFP - ER ( magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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D: Endogenous MOSPD2 (green) staining in HeLa cells expressing the ER marker GFP-ER (magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressing Flag - STARD3NL ( E ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay . Black rectangles indicate the positions of late endosomes ( E ) . Scale bars : 10 µm

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E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressing Flag-STARD3NL (E (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay. Black rectangles indicate the positions of late endosomes (E). Scale bars: 10 µm


E - F : Endogenous MOSPD2 ( green ) staining in HeLa cells expressin Flag - STARD3NL ∆ FFAT ( F ) ( anti - Flag ; magenta ) Data information : ( A , D - F ) : The subpanels on the right are higher magnification ( 3 . 5x ) images of the area outlined in white . The Overlay panel shows merged green and magenta images . The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co - localize are shown in white . Right : Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars : 10

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E-F: Endogenous MOSPD2 (green) staining in HeLa cells expressin Flag-STARD3NL ∆FFAT (F) (anti-Flag; magenta) Data information: (A, D-F): The subpanels on the right are higher magnification (3.5x) images of the area outlined in white. The Overlay panel shows merged green and magenta images. The Coloc panel displays a colocalization mask on which pixels where the green and the magenta channels co-localize are shown in white. Right: Linescan analyses with fluorescence intensities of the green and magenta channels along the white arrow shown on the subpanel Overlay Scale bars: 10 µm


G : Pearson correlation coefficients between endogenous MOSPD2 and Flag - STARD3NL ( WT or ΔFFAT ) staining are shown . Each dot represents a single cell ( number of cells : MOSPD2 - Flag - STARD3NL : 27 ; MOSPD2 - Flag - STARD3NL ΔFFAT : 26 ; from three independent experiments ) . Means and error bars ( SD ) are shown . Mann - Whitney test ( two - tailed P value ; * * * * : P < 0 , 0001 )

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G: Pearson correlation coefficients between endogenous MOSPD2 and Flag-STARD3NL (WT or ΔFFAT) staining are shown. Each dot represents a single cell (number of cells: MOSPD2-Flag-STARD3NL: 27; MOSPD2-Flag-STARD3NL ΔFFAT: 26; from three independent experiments). Means and error bars (SD) are shown. Mann-Whitney test (two-tailed P value; ****: P< 0,0001)


A : Western blot analysis of MOSPD2 , VAP - A and VAP - B proteins in control HeLa cells ( WT ) and in HeLa cells transfected with a control siRNA ( siCtrl ) , and with siRNA targeting MOSPD2 ( siMOSPD2 ) , VAP - A and VAP - B ( siVAP - A / VAP - B ) , and MOSPD2 , VAP - A and VAP - B ( siMOSPD2 / VAP - A / VAP - B )

A: Western blot analysis of MOSPD2, VAP-A and VAP-B proteins in control HeLa cells (WT) and in HeLa cells transfected with a control siRNA (siCtrl), and with siRNA targeting MOSPD2 (siMOSPD2), VAP-A and VAP-B (siVAP-A/VAP-B), and MOSPD2, VAP-A and VAP-B (siMOSPD2/VAP-A/VAP-B)


B - F : TEM images of control HeLa cell ( B : HeLa - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images of control HeLa cell (B: HeLa -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images of control HeLa cell C : HeLa / siCtrl - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images of control HeLa cell C: HeLa/siCtrl -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 ( D : siMOSPD2 - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images o MOSPD2 (D: siMOSPD2 -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o VAP - A and VAP - B ( E : siVAP - A / VAP - B - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images o VAP-A and VAP-B (E: siVAP-A/VAP-B -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


B - F : TEM images o MOSPD2 , VAP - A and VAP - B ( F : siVAP - A / VAP - B / MOSPD2 ) - silenced HeLa cells . An interpretation scheme representing contacts between organelles is shown on the right ; the ER , endosomes and ILV are in dark , light and medium gray , respectively . Mitochondria and Golgi are in pink and light green , respectively . Scale bars : 500 µm

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B-F: TEM images o MOSPD2, VAP-A and VAP-B (F: siVAP-A/VAP-B/MOSPD2) -silenced HeLa cells. An interpretation scheme representing contacts between organelles is shown on the right; the ER, endosomes and ILV are in dark, light and medium gray, respectively. Mitochondria and Golgi are in pink and light green, respectively. Scale bars: 500 µm


G - I : Quantification by stereology of ER - mitochondria ( G contacts . The percentages of mitochondria perimeter in contact with the ER ( G are shown as means and error bars ( SEM ) . ( G ) 200 ( HeLa ) , 208 ( siCtrl ) , 199 ( siMOSPD2 ) , 202 ( siVAP - A / VAP - B ) , 199 ( siMOSPD2 / VAP - A / VAP - B ) mitochondria from 8 , 10 , 8 , 12 and 12 cells , respectively , were

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G-I: Quantification by stereology of ER-mitochondria (G contacts. The percentages of mitochondria perimeter in contact with the ER (G are shown as means and error bars (SEM). (G) 200 (HeLa), 208 (siCtrl), 199 (siMOSPD2), 202 (siVAP-A/VAP-B), 199 (siMOSPD2/VAP-A/VAP-B) mitochondria from 8, 10, 8, 12 and 12 cells, respectively, were analyzed


G - I : Quantification by stereology o ER - endosome ( H contacts The percentages o ) , endosome perimeter in contact with the ER ( H are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o ER-endosome (H contacts The percentages o ), endosome perimeter in contact with the ER (H are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


G - I : Quantification by stereology o endosome - endosome ( I ) contacts . The percentages o endosome perimeter in contact with an endosome ( I ) are shown as means and error bars ( SEM ) ( H , I ) 202 ( HeLa ) , 189 ( siCtrl ) , 148 ( siMOSPD2 ) , 141 ( siVAP - A / VAP - B ) , 221 ( siMOSPD2 / VAP - A / VAP - B ) endosomes from 9 , 19 , 10 , 14 and 12 cells , respectively , were analyzed . Kruskal - Wallis with Dunn " s multiple comparison test ( * : P - values < 0 . 05 ; * * * : P < 0 , 001 ; * * * * : P < 0 . 0001 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G-I: Quantification by stereology o endosome-endosome (I) contacts. The percentages o endosome perimeter in contact with an endosome (I) are shown as means and error bars (SEM) (H, I) 202 (HeLa), 189 (siCtrl), 148 (siMOSPD2), 141 (siVAP-A/VAP-B), 221 (siMOSPD2/VAP-A/VAP-B) endosomes from 9, 19, 10, 14 and 12 cells, respectively, were analyzed. Kruskal-Wallis with Dunn"s multiple comparison test (*: P-values <0.05; ***: P< 0,001; ****: P <0.0001)


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . A ) Immunohistochemistry analysis of small intestinal sections , using the indicated antibodies .

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All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.A) Immunohistochemistry analysis of small intestinal sections, using the indicated antibodies.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . B ) Western blot analysis with protein extracts obtained from ex vivo - purified intestinal crypts and probed with the indicated antibodies .

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All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.B) Western blot analysis with protein extracts obtained from ex vivo-purified intestinal crypts and probed with the indicated antibodies.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . C ) Haematoxylin and eosin staining of sections prepared from different intestinal tracts .

All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.C) Haematoxylin and eosin staining of sections prepared from different intestinal tracts.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . D ) Immunohistochemistry analysis of small intestinal sections , using specific antibodies for the proliferation marker KI67 .

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All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.D) Immunohistochemistry analysis of small intestinal sections, using specific antibodies for the proliferation marker KI67.


All analyses were performed using AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days . E ) Expression analysis by qRT - PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts . Graphs show mean ± SD for three replicates .

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All analyses were performed using AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.E) Expression analysis by qRT-PCR of the proliferation related markers indicated in the figure using RNA extracted from intestinal crypts. Graphs show mean ± SD for three replicates.


A ) PAS and ( B ) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed + / + and AhCre Eedfl / fl mice ( n = 5 ) injected with β - naphthoflavone and sacrificed after 15 days .

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A) PAS and (B) Alcian blue staining of sections prepared from the different intestinal tracts isolated from AhCre Eed+/+ and AhCre Eedfl/fl mice (n=5) injected with β-naphthoflavone and sacrificed after 15 days.


C ) Quantification ( mean ± SD ) of Goblet cells in crypts and villi from the same mice sown in A and B . More than 100 crypts and villi were scored for each condition after 15 days from the first β - naphthoflavone administration .

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C) Quantification (mean ± SD) of Goblet cells in crypts and villi from the same mice sown in A and B. More than 100 crypts and villi were scored for each condition after 15 days from the first β-naphthoflavone administration.


D ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice injected with β - naphthoflavone and sacrificed after 15 days using a lysozyme - specific antibody ( upper panels : LYZ , a Paneth cell - specific marker ) . Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels ( ALPI , an enterocyte - specific marker ) . Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels ( Alcian / KI67 ) .

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D) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice injected with β-naphthoflavone and sacrificed after 15 days using a lysozyme-specific antibody (upper panels: LYZ, a Paneth cell-specific marker). Histochemical assay probing for alkaline phosphatase activity is presented in the middle panels (ALPI, an enterocyte-specific marker). Combined Ki67 immunohistochemistry and histochemical Alcian blue staining is presented in the lower panels (Alcian/KI67).


E ) GFP expression from an Lgr5 - eGFPtransgene identifying LGR5 + ISCs in near - native agarose - embedded sections from Eed + / + and Eed - / - mice at 30 days from the first β - naphthoflavone administration . Cell nuclei were counterstained with DAPI .

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E) GFP expression from an Lgr5-eGFPtransgene identifying LGR5+ ISCs in near-native agarose-embedded sections from Eed+/+ and Eed-/- mice at 30 days from the first β-naphthoflavone administration. Cell nuclei were counterstained with DAPI.


A ) Radiation - induced intestinal regeneration : AhCre Eed + / + and AhCre Eedfl / fl mice were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 4 and 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( n = 6 ) .

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A) Radiation-induced intestinal regeneration: AhCre Eed+/+ and AhCre Eedfl/fl mice were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 4 and 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (n=6).


B ) Immunohistochemistry analyses in AhCre Eed + / + and AhCre Eedfl / fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post - irradiation . C ) Quantification ( mean ± SD ) of KI67 positive cells per crypt ( B ) . More than 100 crypts were scored for each condition .

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B) Immunohistochemistry analyses in AhCre Eed+/+ and AhCre Eedfl/fl mice treated as in A using H3K27me3 and KI67 specific antibodies at 8 days post-irradiation.C) Quantification (mean ± SD) of KI67 positive cells per crypt (B). More than 100 crypts were scored for each condition.


D ) In vitro organoids formation using crypts isolated from AhCre Eed + / + and AhCre Eed - / - mice 15 days after the first β - naphthoflavone injection . Organoids pictures were taken 5 days later at low and higher magnification ( insets ) .

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D) In vitro organoids formation using crypts isolated from AhCre Eed+/+ and AhCre Eed-/- mice 15 days after the first β-naphthoflavone injection. Organoids pictures were taken 5 days later at low and higher magnification (insets).


A ) Volcano plot showing the expression changes with a minimal fold change of 3 ( FC = 3 ) at genome - wide levels in AhCre Eed - / - crypts relative to AhCre Eed + / + crypts 15 days after β - naphthoflavone administration .

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A) Volcano plot showing the expression changes with a minimal fold change of 3 (FC=3) at genome-wide levels in AhCre Eed-/- crypts relative to AhCre Eed+/+ crypts 15 days after β-naphthoflavone administration.


B ) Bar plot showing the percentage of H3K27me3 - positive promoters among the differentially regulated genes in Eed - / - crypts . All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls .

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B) Bar plot showing the percentage of H3K27me3-positive promoters among the differentially regulated genes in Eed-/- crypts. All gene promoters and 5 independent random sampling of 167 gene promoters extracted the mouse genome are presented as specificity controls.


C ) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up - regulated genes with respect to down - regulated genes in Eed - / - crypts .

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C) Intensity profiles for H3K27me3 and SUZ12 occupancy at the promoters of up-regulated genes with respect to down-regulated genes in Eed-/- crypts.


D ) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms .

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D) Genomic snapshots of the Cdkn2a locus showing SUZ12 occupancy and H3K27me3 deposition at the promoter regions of both the p16 and the ARF isoforms.


E ) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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E) Genomic snapshots of the RNAseq profile of the Cdkn2a locus in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


F ) Expression analysis by qRT - PCR ( mean ± SD ) of both Cdkn2a isoforms ( p16Ink4A and p19Arf ) in AhCre Eed + / + and AhCre Eed - / - crypts 15 days after β - naphthoflavone administration .

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F) Expression analysis by qRT-PCR (mean ± SD) of both Cdkn2a isoforms (p16Ink4A and p19Arf) in AhCre Eed+/+ and AhCre Eed-/- crypts 15 days after β-naphthoflavone administration.


G ) Gene set enrichment analysis ( GSEA ) using specific secretory and enterocytic transcriptional signatures with respect to the list of up - regulated genes in Eed - / - crypts .

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G) Gene set enrichment analysis (GSEA) using specific secretory and enterocytic transcriptional signatures with respect to the list of up-regulated genes in Eed-/- crypts.


A ) Haematoxylin and eosin ( top panels ) and PAS staining ( bottom panels ) of sections prepared from different intestinal tracts in AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration ( n = 5 ) .

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A) Haematoxylin and eosin (top panels) and PAS staining (bottom panels) of sections prepared from different intestinal tracts in AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration (n=5).


B ) Quantification ( mean ± SD ) of Goblet cells along the crypt - to - villus axis from the staining shown in A . More than 100 crypts and villi were scored for each condition .

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B) Quantification (mean ± SD) of Goblet cells along the crypt-to-villus axis from the staining shown in A. More than 100 crypts and villi were scored for each condition.


C ) Immunhistochemistry analysis using anti - KI67 antibody of intestinal sections prepared from AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice 15 days after the first β - naphthoflavone administration .

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C) Immunhistochemistry analysis using anti-KI67 antibody of intestinal sections prepared from AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice 15 days after the first β-naphthoflavone administration.


D ) Radiation - induced intestinal regeneration : AhCre Cdkn2a - / - Eed + / + and AhCre Cdkn2a - / - Eedfl / fl mice ( n = 6 ) were injected with β - naphthoflavone , irradiated with 10 Gy 15 days later , and sacrificed 8 days after irradiation . Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin ( first panels ) and Alcian Blue ( last panels ) . Sections were also stained for immunohistochemistry analysis ( second and third panels ) using H3K27me3 and Ki67 - specific antibodies .

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D) Radiation-induced intestinal regeneration: AhCre Cdkn2a-/- Eed+/+ and AhCre Cdkn2a-/- Eedfl/fl mice (n=6) were injected with β-naphthoflavone, irradiated with 10 Gy 15 days later, and sacrificed 8 days after irradiation. Sections prepared from the different intestinal tracts were stained with haematoxylin and eosin (first panels) and Alcian Blue (last panels). Sections were also stained for immunohistochemistry analysis (second and third panels) using H3K27me3 and Ki67-specific antibodies.