A . Representative of Parkin - mediated clearance of mitochondrial outer TOMM20 in HeLa / Parkin treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13 oligos . Scale bars , 20 μm . B . of TOMM20 clearance in experiment shown in ( A ) . Results represent mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons .

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A. Representative images of Parkin-mediated clearance of mitochondrial outer membrane protein TOMM20 in HeLa/Parkin cells treated with indicated siRNA 16 h after treatment with 10 μM CCCP or DMSO vehicle control. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13 oligos. Scale bars, 20 μm.B. Quantification of TOMM20 clearance in the experiment shown in (A). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.


C . Representative of Parkin - mediated clearance of mitochondrial double - stranded ( mtDNA ) in HeLa / HA - Parkin treated with indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin 250 nM antimycin A ( OA ) or DMSO vehicle control . siPEX13 oligo # 1 is shown ; similar results were observed with three other siPEX13 oligos . was masked using DAPI . Scale bars , 20 μm . D . of mtDNA clearance in experiment shown in ( C ) . Results represent box plots of ~ 300 analyzed per sample . Whiskers represent 5 % - 95 % range , each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 ; Kruskal - Wallis H - .

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C. Representative images of Parkin-mediated clearance of mitochondrial double-stranded DNA (mtDNA) in HeLa/HA-Parkin cells treated with indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA) or DMSO vehicle control. siPEX13 oligo #1 is shown; similar results were observed with three other siPEX13 oligos. Nuclear DNA staining was masked using DAPI. Scale bars, 20 μm.D. Quantification of mtDNA clearance in the experiment shown in (C). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001; Kruskal-Wallis H-test.


E . Representative of TOMM20 fragmentation or compaction around perinuclear region in MEFs of indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control . lines indicate borders . Scale bars , 20 μm . F . of of in experiment shown in ( E ) with accumulation of fragmented mitochondria after CCCP treatment . Results represent mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 ; two - unpaired t - .

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E. Representative images of TOMM20 fragmentation or compaction around perinuclear region in MEFs of the indicated genotype 24 h after treatment with 30 μM CCCP or DMSO vehicle control. White lines indicate cell borders. Scale bars, 20 μm.F. Quantification of percentage of cells in experiment shown in (E) with accumulation of fragmented mitochondria after CCCP treatment. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01; two-tailed unpaired t-test.


G . Electron microscopic of E18 . 5 mouse embryonic liver , heart , skeletal muscle . Shown are representative from one mouse . Similar results were observed in three mice per genotype . Arrowheads indicate damaged mitochondria . Scale bars , 1 μm .

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G. Electron microscopic analysis of E18.5 mouse embryonic liver, heart, and skeletal muscle. Shown are representative images from one mouse. Similar results were observed in three mice per genotype. Arrowheads indicate damaged mitochondria. Scale bars, 1 μm.


A . Western blot detection of PEX13 in HeLa / Parkin transfected with indicated siRNA siRNA - resistant PEX13 plasmid . To achieve similar PEX13 expression , 0 . 75 μg PEX13 I326T PEX13 W313G plasmids were transfected compared to 0 . 25 μg WT PEX13 . Total plasmid level was adjusted using empty vector . Asterisk denotes non - specific band .

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A. Western blot detection of PEX13 in HeLa/Parkin cells transfected with indicated siRNA and siRNA-resistant PEX13 plasmid. To achieve similar PEX13 protein expression levels, 0.75 μg PEX13 I326T and PEX13 W313G plasmids were transfected compared to 0.25 μg WT PEX13. Total plasmid level was adjusted using empty vector. Asterisk denotes non-specific band.


B . Representative of Parkin - mediated clearance of TOMM20 in HeLa / Parkin treated with PEX13 siRNA transfected with indicated PEX13 siRNA - resistant plasmid then treated with CCCP ( 10 μM , 16 h ) . Scale bars , 20 μm . See Fig EV3B for representative of mitochondrial ( TOMM20 ) in control treated with DMSO .

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B. Representative images of Parkin-mediated clearance of TOMM20 in HeLa/Parkin cells treated with PEX13 siRNA and transfected with indicated PEX13 siRNA-resistant plasmid and then treated with CCCP (10 μM, 16 h). Scale bars, 20 μm. See Fig EV3B for representative images of mitochondrial morphology (TOMM20 staining) in control cells treated with DMSO.


C . of experiment shown in ( B ) . Results represent mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two - unpaired t - .

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C. Quantification of experiment shown in (B). Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01,***p<0.001; two-tailed unpaired t-test.


D . Western blot detection of PEX13 expression in wild - type ( WT ) PEX13 W313G ( W313G ) mutant human fibroblasts .

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D. Western blot detection of PEX13 expression in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts.


E . Representative of Parkin - mediated clearance of TOMM20 in wild - type ( WT ) PEX13 W313G ( W313G ) mutant human fibroblasts treated with Oligomycin ( 2 . 5 μM ) + Antimycin A ( 250 nM ) for 24 h . Scale bars , 20 μm . W313G varied in ; outlined in upper right panel shows a representative with larger abnormal mitochondrial ( see Fig EV3C for higher resolution imaging ) outlined in lower right panel shows a representative with accumulation of fragmented mitochondria that would be scored as positive in ( F ) . F . of experiment shown in ( E ) . Results represent mean ± of 5 groups of 10 of random fields of ( > 350 analyzed per sample ) . Similar results were observed in two independent experiments . * * * p < 0 . 0001 ; two - unpaired t - .

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E. Representative images of Parkin-mediated clearance of TOMM20 in wild-type (WT) and PEX13 W313G (W313G) mutant primary human fibroblasts treated with Oligomycin (2.5 μM) + Antimycin A (250 nM) for 24 h. Scale bars, 20 μm. W313G cells varied in morphology; outlined cell in upper right panel shows a representative cell with larger size and abnormal mitochondrial morphology (see Fig EV3C for higher resolution imaging) and outlined cell in lower right panel shows a representative cell with accumulation of fragmented mitochondria that would be scored as positive in (F).F. Quantification of experiment shown in (E). Results represent mean ± SEM of 5 groups of 10 images of random fields of cells (>350 cells analyzed per sample). Similar results were observed in two independent experiments. ***p<0.0001; two-tailed unpaired t-test.


A - C Representative of PEX13 PMP70 ( A ) , PEX13 TOMM20 ( B ) , or PEX13 - Flag WIPI2 ( C ) colocalization in HeLa / Parkin transfected with PEX13 after 4 h DMSO or CCCP treatment . Scale bars , 20 μm ( A - B ) 5 μm ( C ) .

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A-C Representative images of PEX13 and PMP70 (A), PEX13 and TOMM20 (B), or PEX13-Flag and WIPI2 (C) colocalization in HeLa/Parkin cells transfected with PEX13 after 4 h DMSO or CCCP treatment. Scale bars, 20 μm (A-B) and 5 μm (C).


D Western blot detection of endogenous PEX13 in HeLa / Parkin treated with 10 μM CCCP for indicated time .

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D Western blot detection of endogenous PEX13 in HeLa/Parkin cells treated with 10 μM CCCP for the indicated time.


A . Quantitative real - time PCR detection of mRNA for PEX3 , PEX13 , PEX14 , PEX19 in HeLa transfected with indicated siRNA . * * * p < 0 . 001 ; two - unpaired t - .

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A. Quantitative real-time PCR detection of mRNA levels for PEX3, PEX13, PEX14, and PEX19 in HeLa cells transfected with the indicated siRNA. ***p<0.001; two-tailed unpaired t-test.


B . of Parkin - mediated TOMM20 clearance in HeLa / Parkin transfected with indicated siRNA 16 h after treatment with 10 µM CCCP . Results represent mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons .

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B. Quantification of Parkin-mediated TOMM20 clearance in HeLa/Parkin cells transfected with the indicated siRNA 16 h after treatment with 10 µM CCCP. Results represent mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. **p<0.01, ***p<0.001; one-way ANOVA with adjustment for multiple comparisons.


C . of mtDNA clearance in HeLa / HA - Parkin transfected with indicated siRNA 8 h after treatment with 2 . 5 μM oligomycin 250 nM antimycin A ( OA ) . Results represent box plots of ~ 300 analyzed per sample . Whiskers represent 5 % - 95 % range , each outlier is represented by a dot . Similar results were observed in three independent experiments . * * * * p < 0 . 0001 , NS = not significant ; Kruskal - Wallis H - .

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C. Quantification of mtDNA clearance in HeLa/HA-Parkin cells transfected with the indicated siRNA 8 h after treatment with 2.5 μM oligomycin and 250 nM antimycin A (OA). Results represent box plots of ~300 cells analyzed per sample. Whiskers represent 5%-95% range, and each outlier is represented by a dot. Similar results were observed in three independent experiments. ****p<0.0001, NS= not significant; Kruskal-Wallis H-test.


D . of GFP - LC3 in HeLa / GFP - LC3 treated with 10 nM Baf A1 or DMSO vehicle cultured in normal medium or 3 h HBSS starvation media . Results represent mean ± in triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , NS = not significant ; one - way ANOVA with adjustment for multiple comparisons . Statistical analyses refer to differences between Pex or ATG7 siRNAs vs . NC siRNA within each treatment group .

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D. Quantification of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or 3 h HBSS starvation media. Results represent mean ± SEM in triplicate samples (~100 cells analyzed per sample). Similar results were observed in three independent experiments. *p<0.05, **p<0.01, ***p<0.001, NS= not significant; one-way ANOVA with adjustment for multiple comparisons. Statistical analyses refer to the differences between Pex or ATG7 siRNAs vs. NC siRNA within each treatment group.


( A ) Hematoxylin Eosin ( H & E ) of WT K14ΔNLef1 HF from back skin at 11 . 5 ( early anagen ) . arrowheads : ectopic SG epidermal cysts . Data information Scale bars : 50 μm .

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(A) Hematoxylin and Eosin (H&E) staining of WT and K14ΔNLef1 HF from back skin at 11.5 weeks (early anagen). White arrowheads: ectopic SG and epidermal cysts. Data information Scale bars: 50 μm.


( B ) Tail epidermal whole - mounts from WT K14ΔNLef1 mice labelled with Fabp5 , Krt15 counterstained with Dapi . Data information : Scale bars : 50 μm .

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(B) Tail epidermal whole-mounts from WT and K14ΔNLef1 mice labelled with Fabp5, Krt15 and counterstained with Dapi. Data information: Scale bars: 50 μm.


( C ) Average signal of ΔNLef1 binding sites detected by a Lef1 antibody ( red ) as compared to input alone ( grey ) negative control ( Neg . Ctl . , black ) . motif revealed Lef1 classical consensus . Representative plots of ChIP - Seq reads aligned to Vgll4 , Runx1 Gata6 loci .

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(C) Average signal intensity of ΔNLef1 binding sites detected by a Lef1 antibody (red) as compared to input alone (grey) and negative control (Neg. Ctl., black). DNA motif analysis revealed Lef1 classical consensus sequence. Representative plots of ChIP-Seq reads aligned to the Vgll4, Runx1 and Gata6 loci.


( D ) Heat - map ( Pearson correlation ) of differentially expressed genes ( DEG ) between different micro - dissected regions ( IFE , HF SG ) from WT mice ( left panel ) ΔNLef1 Chip - seq data show ΔNLef1 direct transcriptional targets in black ( right panel ) .

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(D) Heat-map (Pearson correlation) of differentially expressed genes (DEG) between different micro-dissected regions (IFE, HF and SG) from WT mice (left panel) ΔNLef1 Chip-seq data show ΔNLef1 direct transcriptional targets in black (right panel).


( E ) Gene Set Enrichment comparing gene location of ΔNLef1 peaks SG gene expression signature .

(E) Gene Set Enrichment Analysis comparing gene location of ΔNLef1 peaks and SG gene expression signature.


( F ) RT - qPCR of Gata6 in WT K14ΔNLef1 keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled ( scr . ) . Data are means ± of three independent wells . Data information Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 .

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(F) RT-qPCR analysis of Gata6 in WT and K14ΔNLef1 primary keratinocytes transfected with siRNA targeting ΔNLef1 or a scrambled sequence (scr.). Data are means ± SEM of three independent wells. Data information Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.


( G ) RT - qPCR of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1 - or ΔN34Lef1 - expressing plasmid . ∆ N34Lef1 is human ortholog of murine ∆ N32Lef1 , which is expressed in K14 ∆ NLef1 transgenic mice Data are means ± of three independent experiments . Data information : Statistical analyses were performed with an ordinary one - way ANOVA : ( ns ) not significant ; ( * ) p - value < 0 . 05 ; ( * * * ) p - value < 0 . 0005 .

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(G) RT-qPCR analysis of Gata6 in human SebE6E7 sebocytes transfected for 48h with a mock plasmid or a Lef1- or ΔN34Lef1-expressing plasmid. ∆N34Lef1 is the human ortholog of murine ∆N32Lef1, which is expressed in K14∆NLef1 transgenic mice Data are means ± SEM of three independent experiments. Data information: Statistical analyses were performed with an ordinary one-way ANOVA: (ns) not significant; (*) p-value < 0.05; (***) p-value < 0.0005.


( H ) Dorsal skin sections of WT K14ΔNLef1 mice stained for Krt14 Gata6 . Data information Scale bars : 50 μm .

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(H) Dorsal skin sections of WT and K14ΔNLef1 mice stained for Krt14 and Gata6. Data information Scale bars: 50 μm.


( I ) Heat - maps of DEG from WT - or K14ΔNLef1 - unfractionated ( ) or sorted ( Itga6 + Cd34 - , basal ; Itga6 + Cd34 + , HFSC ) keratinocytes in comparison with IFE , SG , HF gene signatures ranked from high to low expression .

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(I) Heat-maps of DEG from WT- or K14ΔNLef1-unfractionated (all) or sorted (Itga6+Cd34-, basal; Itga6+Cd34+, HFSC) keratinocytes in comparison with IFE, SG, and HF gene signatures ranked from high to low expression.


( J ) Heat - map depicting similarities , as Pearson ' s correlation coefficient , between DEG from keratinocytes from WT or K14ΔNLef1 mice IFE vs SG vs HF transcriptome ( left panel ) . Additional correlation with ΔNLef1 Chip - seq data is depicted in right heat - map .

(J) Heat-map depicting similarities, as Pearson's correlation coefficient, between DEG from keratinocytes from WT or K14ΔNLef1 mice and IFE vs SG vs HF transcriptome analysis (left panel). Additional correlation with ΔNLef1 Chip-seq data is depicted in the right heat-map.


( A ) Sections of WT embryonic skin at different HF stages stained for Gata6 , Lef1 , Sox9 counterstained with Dapi . Black asterisks indicate overexposed areas of nonspecific Lef1 in suprabasal epidermis . of of labelled for both Gata6 Lef1 , or Gata6 Sox9 in stages 4 - 5 HF are shown ( upper right panel ) . Data are means ± SD were obtained from 9 HF from 3 mice . ( B ) Sections of E18 . 5 WT Lef1 - / - mouse skin stained for Lef1 Gata6 . Deletion of Lef1 does not impair Gata6 expression . Data information : Scale bars : 50 μm .

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(A) Sections of WT embryonic skin at different HF stages stained for Gata6, Lef1, Sox9 and counterstained with Dapi. Black asterisks indicate overexposed areas of nonspecific Lef1 staining in the suprabasal epidermis. Quantification of the percentage of cells labelled for both Gata6 and Lef1, or Gata6 and Sox9 in stages 4-5 HF are shown (upper right panel). Data are means ± SD and were obtained from 9 HF from 3 mice. (B) Sections of E18.5 WT and Lef1-/- mouse skin stained for Lef1 and Gata6. Deletion of Lef1 does not impair Gata6 expression. Data information: Scale bars: 50 μm.


( C ) Sections of WT K14ΔNβcateninER ( K14ΔNβ - catER D2 D4 strains ) adult dorsal skin stained with antibodies against Krt14 Gata6 . Topical treatment with 1 or 6 doses of 4OHT activates β - catenin leading to anagen induction ectopic hair follicles but not Gata6 expression . Data information : Scale bars : 50 μm .

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(C) Sections of WT and K14ΔNβcateninER (K14ΔNβ-catER D2 and D4 strains) adult dorsal skin stained with antibodies against Krt14 and Gata6. Topical treatment with 1 or 6 doses of 4OHT activates β-catenin leading to anagen induction and ectopic hair follicles but not Gata6 expression. Data information: Scale bars: 50 μm.


( D ) Sections of E18 . 5 WT K14Cre / βCat Flox ( ex3 ) / + mouse skin stained for Sox9 Gata6 . Activation of Wnt / β - catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of HFSC marker Sox9 . Data information : Scale bar : 25 μm .

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(D) Sections of E18.5 WT and K14Cre/βCat Flox(ex3)/+ mouse skin stained for Sox9 and Gata6. Activation of Wnt/β-catenin signaling during epidermis development does not induce Gata6 expression but results in ectopic expression of the HFSC marker Sox9. Data information: Scale bar: 25 μm.


( A ) Bright - field of whole - mount WT Gata6 cKO epidermis , showing abnormal SG upon loss of Gata6 . of hypotrophic SG in WT , heterozygous ( Het ) Gata6 cKO epidermal whole - mounts . Data are boxplots with indication of means . Box limits are minimum maximum values . An average of 165 HF per mouse ( from 3 to 4 mice per genotype ) was analyzed . ( * ) p - value < 0 . 05 ; ( * * ) p - value < 0 . 005 ; unpaired Student ' s t - . Data information : Scale bars : 50 μm .

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(A) Bright-field images of whole-mount WT and Gata6 cKO epidermis, showing abnormal SG upon loss of Gata6. Quantification of hypotrophic SG in WT, heterozygous (Het) and Gata6 cKO epidermal whole-mounts. Data are boxplots with indication of means. Box limits are minimum and maximum values. An average of 165 HF per mouse (from 3 to 4 mice per genotype) was analyzed. (*) p-value < 0.05; (**) p-value < 0.005; unpaired Student's t-test. Data information: Scale bars: 50 μm.


( B ) Schematic representation of plasmid constructs used for in utero lentiviral infection . Whole - mounts or sections of adult infected epidermis with empty vector ( EV ) or Gata6 - ires - GFP ( G6OE ) lentivirus were stained for GFP Fasn . In vivo overexpression of Gata6 leads to ectopic Fasn expression in HF / SG unit . dotted lines define SG , yellow dotted lines define a cyst . Note that cyst is mostly negative for Fasn in agreement with its SD - like phenotype . arrows indicate GFP - positive infected . These are stained with Fasn only in G6OE condition . H & E - stained skin from G6OE mice shows a cyst with SG elements in HF unit ( black arrows ) . for Gata6 ( both endogenous exogenous ) shows that Gata6 expression occurs in a limited number of ( representative image in upper right panel ) . Bottom left graph shows of of clones labelled for both Fasn GFP in SG , HF IFE compartments . Data are means ± SD originate from 4 EV mice 8 G6OE mice ( average of 11 clones per mouse ) . ( C ) Whole - mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX . Ectopic Gata6 are not stained with LipidTOX indicating an incomplete sebaceous maturation . Data information : Scale bars : 50 μm .

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(B) Schematic representation of plasmid constructs used for in utero lentiviral infection. Whole-mounts or sections of adult infected epidermis with empty vector (EV) or Gata6-ires-GFP (G6OE) lentivirus were stained for GFP and Fasn. In vivo overexpression of Gata6 leads to ectopic Fasn expression in the HF/SG unit. White dotted lines define SG, and yellow dotted lines define a cyst. Note that the cyst is mostly negative for Fasn in agreement with its SD-like phenotype. White arrows indicate GFP-positive infected cells. These cells are stained with Fasn only in G6OE condition. H&E-stained skin from G6OE mice shows a cyst with SG elements in the HF unit (black arrows). Staining for Gata6 (both endogenous and exogenous) shows that Gata6 expression occurs in a limited number of cells (representative image in upper right panel). Bottom left graph shows quantification of the percentage of clones labelled for both Fasn and GFP in the SG, HF and IFE compartments. Data are means ± SD and originate from 4 EV mice and 8 G6OE mice (average of 11 clones per mouse). (C) Whole-mounts adult infected epidermis with EV or G6OE lentivirus stained with LipidTOX. Ectopic Gata6 cells are not stained with LipidTOX indicating an incomplete sebaceous maturation. Data information: Scale bars: 50 μm.


( D ) Lineage tracing experiments in Gata6EGFPCreERT2 : Rosa26 - fl / STOP / fl - tdTomato ( Gata6creER ROSA - dTom ) mice . A single dose of 4OHT was injected in pregnant at E16 . 5 . Tail skin from pups was collected at P13 . Representative example of whole - mount epidermis showing tdTomato labelled counterstained with Dapi ( top left panel ) . Right panels show different Z - stacks related to this whole - mount . Gata6 progenies are mainly found in upper SG / JZ . Localization of Gata6 progenies ( dTomato + ) was quantified in 20 - 26 pilosebaceous units per mouse ( N = 4 mice ) ( bottom left panel ) . Data are means ± SD . As a control , was also performed in Rosa26 - fl / STOP / fl - tdTomato ( ROSA - dTom ) mice that were treated similarly to Gata6creER ROSA - dTomato mice . Data information : Scale bar : 25 μm .

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(D) Lineage tracing experiments in Gata6EGFPCreERT2:Rosa26-fl/STOP/fl-tdTomato (Gata6creER ROSA-dTom) mice. A single dose of 4OHT was injected in pregnant females at E16.5. Tail skin from pups was collected at P13. Representative example of whole-mount epidermis showing tdTomato labelled cells counterstained with Dapi (top left panel). Right panels show the different Z-stacks related to this whole-mount. Gata6 progenies are mainly found in the upper SG/JZ. Localization of Gata6 progenies (dTomato+) was quantified in 20-26 pilosebaceous units per mouse (N=4 mice) (bottom left panel). Data are means ± SD. As a control, quantification was also performed in Rosa26-fl/STOP/fl-tdTomato (ROSA-dTom) mice that were treated similarly to Gata6creER ROSA-dTomato mice. Data information: Scale bar: 25 μm.


( A ) Sections of mouse skin tumors were stained with antibodies to Gata6 Krt14 . A papilloma found in WT mice treated with DMBA TPA ( left panel ) , is compared with sebaceous tumors found in K14ΔNLef1 mice ( right panels ) . Data information : Scale bars : 100 μm .

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(A) Sections of mouse skin tumors were stained with antibodies to Gata6 and Krt14. A papilloma tumor found in WT mice treated with DMBA and TPA (left panel), is compared with sebaceous tumors found in K14ΔNLef1 mice (right panels). Data information: Scale bars: 100 μm.


( B ) burden incidence in DMBA - treated K14ΔNLef1 ( n = 11 ) K14ΔNLef1 : cKO ( n = 8 ) mice . ( * * * ) p - value < 0 . 001 ; Wilcoxon matched - pairs signed rank . Data information : Data are means ± .

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(B) Tumor burden and tumor incidence in DMBA-treated K14ΔNLef1 (n=11) and K14ΔNLef1:cKO (n=8) mice. (***) p-value < 0.001; Wilcoxon matched-pairs signed rank test. Data information: Data are means ± SEM.


( C ) Representative H & E - stained tumors of K14ΔNLef1 K14ΔNLef1 : cKO mice with of each type relative to total number of tumors in each group ( middle panel ) as average absolute number of tumors per mice ( right panel ) . A total of 268 tumors were analyzed . 143 tumors were found in 7 K14ΔNLef1 mice ( 103 sebaceous tumors 40 papillomas ) , while 125 were found in 5 K14ΔNLef1 : cKO mice ( 55 sebaceous tumors 70 papillomas ) . ( * * ) p - value < 0 . 005 , Student ' s t - . Data information : ( C ) Scale bar : 1 mm .

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(C) Representative H&E-stained tumors of K14ΔNLef1 and K14ΔNLef1:cKO mice with quantification of each tumor type relative to the total number of tumors in each group (middle panel) and as the average absolute number of tumors per mice (right panel). A total of 268 tumors were analyzed. 143 tumors were found in 7 K14ΔNLef1 mice (103 sebaceous tumors and 40 papillomas), while 125 were found in 5 K14ΔNLef1:cKO mice (55 sebaceous tumors and 70 papillomas). (**) p-value < 0.005, Student's t-test. Data information: (C) Scale bar: 1 mm.


( D ) Representative of mouse skin sections labelled with antibodies to Plet1 Atp6v1c2 . Data information : D ) Scale bars : 100 μm .

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(D) Representative images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2. Data information: D) Scale bars: 100 μm.


( A ) Heat - map highlighting differentially expressed genes ( DEG ) between WT Gata6 cKO keratinocytes Gata6 direct transcriptional targets from Chip - Seq data are shown in grey .

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(A) Heat-map highlighting differentially expressed genes (DEG) between WT and Gata6 cKO keratinocytes Gata6 direct transcriptional targets from Chip-Seq data are shown in dark grey.


( C ) MSI performed on skin tumors from DMBA - treated K14ΔNLef1 K14ΔNLef1 : cKO mice ( 5 mice in each group ) . Representative microsatellite profiles of K14ΔNLef1 K14ΔNLef1 : cKO tumors . Peak heights were normalized to highest peak in each microsatellite profile to obtain relative frequency of each allele for 5 different markers . " 0 " indicates position of highest peak in bp . ( * ) p - value < 0 . 05 ; ( * * ) p - value < 0 . 001 ; paired t - .

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(C) MSI analysis performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice (5 mice in each group). Representative microsatellite profiles of K14ΔNLef1 and K14ΔNLef1:cKO tumors. Peak heights were normalized to the highest peak in each microsatellite profile to obtain the relative frequency of each allele for 5 different markers. "0" indicates the position of the highest peak in bp. (*) p-value < 0.05; (**) p-value < 0.001; paired t-test.


( D ) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA - treated K14ΔNLef1 K14ΔNLef1 : cKO mice . 4 mice were included in each group . A technical control is displayed ( without antibody incubation ) . Semi - quantitative of Mlh1 statistical ( χ2 ) were performed . Data information : Scale bar : 250 μm .

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(D) Representative immunohistochemistry stainings against Mlh1 performed on skin tumors from DMBA-treated K14ΔNLef1 and K14ΔNLef1:cKO mice. 4 mice were included in each group. A technical control is displayed (without primary antibody incubation). Semi-quantitative analysis of Mlh1 staining and statistical analysis (χ2 test) were performed. Data information: Scale bar: 250 μm.


( E ) Representative immunohistochemistry stainings against Msh2 semi - quantitative Data information : Scale bar : 250 μm .

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(E) Representative immunohistochemistry stainings against Msh2 and semi-quantitative analysis Data information: Scale bar: 250 μm.


( A ) Normal human scalp skin sections stained with Gata6 PanKeratin antibodies . Endogenous Gata6 expression is found in JZ , upper SG SD . As in mouse skin , Gata6 is not expressed in sweat ( SwG ) . ( B ) Representative sections of human skin tumors stained with Gata6 PanKeratin antibodies . of of Gata6 - positive tumors within each histopathological group ( middle left panel ) of of Gata6 - positive within each positive sample ( middle right panel ) . Note that least positive sample showed about 3 % of Gata6 - positive . was performed on 73 different samples . SSC : squamous carcinoma , SCAP : syringocystadenoma papilliferum , EMPD : extramammary Paget ' s disease , nod . / superf . / scler . BCC : nodular / superficial / sclerodermiform basal carcinoma , Steatocystoma M . : steatocystoma multiplex , FSCH : folliculosebaceous cystic hamartoma . Heat - map displaying - log ( p - value ) of χ2 computed between different types ( bottom left panel ) . ( C ) Gata6 expression ( measured in Transcripts Per Million , TPM ) in human SCC ( n = 6 ) , BCC ( n = 6 ) sebaceous carcinoma ( n = 14 ) samples from ( North et al , 2018 ) . Data are means ± SD . ( * * ) p - value ≤ 0 . 005 , Student ' s t - . Data information : ( A B ) Scale bars : 100 μm .

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(A) Normal human scalp skin sections stained with Gata6 and PanKeratin antibodies. Endogenous Gata6 expression is found in the JZ, upper SG and SD. As in mouse skin, Gata6 is not expressed in sweat glands (SwG). (B) Representative sections of human skin tumors stained with Gata6 and PanKeratin antibodies. Quantification of the percentage of Gata6-positive tumors within each histopathological group (middle left panel) and quantification of the percentage of Gata6-positive cells within each positive sample (middle right panel). Note that the least positive sample showed about 3% of Gata6-positive cells. The analysis was performed on 73 different tumor samples. SSC : squamous cell carcinoma, SCAP: syringocystadenoma papilliferum, EMPD: extramammary Paget's disease, nod./superf./scler. BCC: nodular/ superficial/ sclerodermiform basal cell carcinoma, Steatocystoma M.: steatocystoma multiplex, FSCH: folliculosebaceous cystic hamartoma. Heat-map displaying the -log(p-value) of χ2 test computed between the different tumor types (bottom left panel). (C) Gata6 expression (measured in Transcripts Per Million, TPM) in human SCC (n=6), BCC (n=6) and sebaceous carcinoma (n=14) samples from (North et al, 2018). Data are means ± SD. (**) p-value ≤ 0.005, Student's t-test. Data information: (A and B) Scale bars: 100 μm.


( A ) Differentiation status ( poor , moderate or well - differentiated ) , circumscription ( not infiltrative , focally infiltrative or infiltrative ) , log10 total number of mutations Gata6 mutations in 32 human sebaceous carcinomas ( SebC ) separated in 3 subgroups : pauci - mutational , MSI - related UV - related For Gata6 mutations , most deleterious mutation is displayed . n . a . : not assessed .

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(A) Differentiation status (poor, moderate or well-differentiated), circumscription (not infiltrative, focally infiltrative or infiltrative), log10 total number of mutations and Gata6 mutations in 32 human sebaceous carcinomas (SebC) separated in 3 subgroups: pauci-mutational, MSI-related and UV-related For Gata6 mutations, the most deleterious mutation is displayed. n.a.: not assessed.


( C ) Gata6 expression ( measured in Transcripts Per Million , TPM ) in human pauci - mutational ( pauci - mut ) SebC ( n = 4 ) , MSI - related SebC ( n = 4 ) , UV - related SebC ( n = 5 ) samples . Data are means ± SD . ( * ) p - value ≤ 0 . 05 , Student ' s t - .

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(C) Gata6 expression (measured in Transcripts Per Million, TPM) in human pauci-mutational (pauci-mut) SebC (n=4), MSI-related SebC (n=4), UV-related SebC (n=5) samples. Data are means ± SD. (*) p-value ≤ 0.05, Student's t-test.


( D - F ) Graphs showing number of mutations per megabase ( Mutations / Mb ) ( D ) , number of Somatic Single - Nucleotide Variants ( SSNV ) ( E ) indel ( F ) of Gata6mut ( n = 3 ) or Gata6wt ( n = 6 ) MSI - related SebC ( left panels ) of Gata6mut ( n = 3 ) or Gata6wt ( n = 7 ) UV - related SebC ( right panels ) . Data are means ± SD .

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(D-F) Graphs showing the number of mutations per megabase (Mutations/Mb) (D), the number of Somatic Single-Nucleotide Variants (SSNV) (E) and indel (F) of Gata6mut (n=3) or Gata6wt (n=6) MSI-related SebC (left panels) and of Gata6mut (n=3) or Gata6wt (n=7) UV-related SebC (right panels). Data are means ± SD.


( G ) Heat - map displaying mean expression level of MMR genes ( measured in TPM ) in Gata6wt ( n = 10 ) Gata6mut ( n = 3 ) SebC .

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(G) Heat-map displaying the mean expression level of MMR genes (measured in TPM) in Gata6wt (n=10) and Gata6mut (n=3) SebC.


( a ) Hybridization of from 90 HapMap YRI samples to Affymetrix SNP 6 . 0 array reveals a correlated pattern of variation in across six copy - number probes spanning region upstream of IRGM , suggesting existence of a common copy - number polymorphism . ( Darker shades of red represent reduced hybridization . ) Quantitative PCR indicated that copy - number - variable region was an insertion / deletion ( Supplementary Fig . 1 ) .

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(a) Hybridization of DNA from 90 HapMap YRI samples to the Affymetrix SNP 6.0 array reveals a correlated pattern of variation in intensity across six copy-number probes spanning the region upstream of IRGM, suggesting the existence of a common copy-number polymorphism. (Darker shades of red represent reduced hybridization intensity.) Quantitative PCR indicated that the copy-number-variable region was an insertion/deletion (Supplementary Fig. 1).


( b ) Mapping of deletion breakpoints by microarray PCR . Red indicates evidence for deletion ; black indicates evidence to contrary ; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele .

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(b) Mapping of the deletion breakpoints by microarray and PCR. Red indicates evidence for deletion; black indicates evidence to the contrary; blue arrows indicate locations of flanking PCR primers able to generate a PCR product in individuals with at least one deletion allele.


( d ) CD association of typed imputed polymorphisms in IRGM region in NIDDK IBDGC genome scan . Blue trace indicates recombination map . deletion polymorphism rs13361189 are indicated by red arrows . rs13361189 ( which is in perfect linkage disequilibrium with deletion , r2 = 1 . 0 ) was also most strongly associated SNP in combined WTCCC genome scan replication study .

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(d) CD association of typed and imputed polymorphisms in the IRGM region in the NIDDK IBDGC genome scan. Blue trace indicates recombination map. The deletion polymorphism and rs13361189 are indicated by red arrows. rs13361189 (which is in perfect linkage disequilibrium with the deletion, r2 = 1.0) was also the most strongly associated SNP in the combined WTCCC genome scan and replication study.


( a ) exonic SNP rs10065172 , which is in strong linkage disequilibrium with 20 - kb insertion / deletion polymorphism upstream of IRGM , can be used to distinguish IRGM transcripts that arise from deletion haplotype from IRGM transcripts that arise from reference haplotype . This makes it possible to measure relative expression of two haplotypes in heterozygotes by measuring relative abundance of two rs10065172 alleles in cDNA . ( Yellow green rectangles indicate transcribed ; green rectangles indicate - coding . ) ( b - g ) Differential expression of IRGM from two structural haplotypes in heterozygotes . relative abundance of C T alleles of rs10065172 in cDNA ( colored circles ) genomic ( black squares ) from human tissues lines were measured . Gray squares indicate control measurements for genomic from 48 HapMap YRI individuals , identifying three reference genotype classes CC , CT TT . Genomic from other samples was heterozygous for C T alleles ( black squares ) ; HeLa genomic ( b , c ) showed a 2 : 1 allelic ( CCT ) reflecting HeLa ' s triploid 5q karyotypic status . In e , cDNA genomic from lymphoblastoid lines from ten different heterozygous individuals were analyzed : ten different colors represent ten individuals ; technical replicates from same are connected by segments . HeLa , SNU182 lymphoblastoid lines expressed C allele of rs10065172 more strongly than T allele ( b - e ) ; HCT116 bronchial expressed T allele much more strongly than C allele ( f , g ) .

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(a) The exonic SNP rs10065172, which is in strong linkage disequilibrium with the 20-kb insertion/deletion polymorphism upstream of IRGM, can be used to distinguish IRGM transcripts that arise from the deletion haplotype from IRGM transcripts that arise from the reference haplotype. This makes it possible to measure the relative expression of the two haplotypes in heterozygotes by measuring the relative abundance of the two rs10065172 alleles in cDNA. (Yellow and green rectangles indicate transcribed sequence; green rectangles indicate protein-coding sequence.) (b-g) Differential expression of IRGM from the two structural haplotypes in heterozygotes. The relative abundance of the C and T alleles of rs10065172 in cDNA (colored circles) and genomic DNA (black squares) from human tissues and cell lines were measured. Gray squares indicate control measurements for genomic DNA from 48 HapMap YRI individuals, identifying the three reference genotype classes CC, CT and TT. Genomic DNA from all other samples was heterozygous for the C and T alleles (black squares); HeLa genomic DNA (b,c) showed a 2:1 allelic ratio (CCT) reflecting HeLa's triploid 5q karyotypic status. In e, cDNA and genomic DNA from lymphoblastoid cell lines from ten different heterozygous individuals were analyzed: the ten different colors represent the ten individuals; technical replicates from the same cell line are connected by line segments. HeLa, SNU182 and the lymphoblastoid cell lines expressed the C allele of rs10065172 more strongly than the T allele (b-e); HCT116 and primary bronchial cells expressed the T allele much more strongly than the C allele (f,g).


( a ) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six - to eightfold in HeLa . were transfected with control ( siControl ) or IRGM - targeted ( siIRGM1 , siIRGM2 ) siRNA duplexes assayed 48 h later by quantitative RT - PCR . Error bars , s . d .

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(a) siRNA constructs directed at IRGM reduced endogenous IRGM transcripts by six- to eightfold in HeLa cells. Cells were transfected with control (siControl) or IRGM-targeted (siIRGM1, siIRGM2) siRNA duplexes and assayed 48 h later by quantitative RT-PCR. Error bars, s.d.


( b ) IRGM knockdown reduces efficiency of anti - autophagy . HeLa stably expressing LC3 - GFP ( a marker for autophagic vesicles ) were transfected with control or IRGM - directed siRNA oligos , then infected with S . typhimurium after 48 h . of encapsulated in LC3 - positive vesicles was determined by microscopy . Reductions in IRGM expression resulted in a significant loss of anti - autophagy compared to control .

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(b) IRGM knockdown reduces the efficiency of anti-bacterial autophagy. HeLa cells stably expressing LC3-GFP (a marker for autophagic vesicles) were transfected with control or IRGM-directed siRNA oligos, then infected with S. typhimurium after 48 h. The percentage of bacteria encapsulated in LC3-positive vesicles was determined by microscopy. Reductions in IRGM expression resulted in a significant loss of anti-bacterial autophagy compared to control cells.


( c ) Microscopic examination reveals a decreased number of autophagically encapsulated in IRGM knockdown . Control siRNA - treated ( siControl ) IRGM siRNA - treated ( siIRGM1 , siIRGM2 ) were infected with S . typhimurium SL1344 imaged by confocal microscopy . Control show numerous ( red in merged image ) surrounded by LC3 - GFP membranes ( green in merged image ) . Such - containing autophagosomes ( indicated by dashed boxes ) were almost completely absent in IRGM - deficient . actin cytoskeleton was visualized using phalloidin ( blue in merge ) . are flat projections of confocal z - stacks . Scale bars , 10 μm . (

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(c) Microscopic examination reveals a decreased number of autophagically encapsulated bacteria in IRGM knockdown cells. Control siRNA-treated (siControl) and IRGM siRNA-treated (siIRGM1, siIRGM2) cells were infected with S. typhimurium SL1344 and imaged by confocal microscopy. Control cells show numerous bacteria (red in merged image) surrounded by LC3-GFP membranes (green in merged image). Such bacteria-containing autophagosomes (indicated by dashed boxes) were almost completely absent in IRGM-deficient cells. The actin cytoskeleton was visualized using phalloidin (blue in merge). Images are flat projections of confocal z-stacks. Scale bars, 10 μm. (


( d ) HeLa stably expressing LC3 - GFP were transfected with increasing amounts of an IRGM expression construct infected 24 h later with S . typhimurium . of found within LC3 - positive vesicles increased with increasing amounts of exogenous IRGM . expression were confirmed by blotting lysates for Flag - IRGM expression , using an antibody to Flag to detect exogenous IRGM actin as a loading control ( lower panel ) . Error bars in b d , s . e . m .

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(d) HeLa cells stably expressing LC3-GFP were transfected with increasing amounts of an IRGM expression construct and infected 24 h later with S. typhimurium. The percentage of bacteria found within LC3-positive vesicles increased with increasing amounts of exogenous IRGM. Protein expression levels were confirmed by blotting lysates for Flag-IRGM expression, using an antibody to Flag to detect exogenous IRGM and actin as a loading control (lower panel). Error bars in b and d, s.e.m.


B . C . Molecular models on crystal structures previously published [ 44 ] represent configurations of two dimers on : Sox2 - Oct4 heterodimer ( B ) Oct6 - Oct6 homodimer ( C ) . Oct4 POU domain , green ; Oct4 / Oct6 linker region , magenta ; Sox2 HMG , cyan ; Oct6 POU domain , orange . helixes of POU domains are numbered . In both models , is represented as a grey surface . Unless indicated otherwise , representation is kept throughout manuscript .

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B. and C. Molecular models based on the crystal structures previously published [44] represent configurations of two dimers on DNA: Sox2-Oct4 heterodimer (B) and Oct6-Oct6 homodimer (C). Oct4 POU domain, green; Oct4/Oct6 linker region, magenta; Sox2 HMG, cyan; Oct6 POU domain, orange. Individual helixes of POU domains are numbered. In both models, the DNA is represented as a light grey surface. Unless indicated otherwise, the representation is kept throughout the manuscript.


D . EMSAs with a 2 - fold dilutions series starting at 2 μM of Oct4 POU ( left ) or Oct6 POU ( right ) in presence of cy5 - labeled MORE . free bound as monomer or dimer are indicated .

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D. EMSAs with a 2-fold dilutions series starting at 2 μM of the Oct4 POU (left) or the Oct6 POU (right) in the presence of cy5-labeled MORE DNA. The free DNA and DNA bound as monomer or dimer are indicated.


E . F . Bar plots of EMSA - derived cooperativity factors for Oct - Oct homodimers on MORE ( E ) or Sox2 - Oct heterodimers on SoxOct element ( F ) are shown for a panel of six POU . mean is shown with standard deviation as error bars ( n ≥ 3 ) Tukey multiple comparison of means was used for assessment of statistical significances ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ) . N . A . : not assessed .

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E. and F. Bar plots of EMSA-derived cooperativity factors for Oct-Oct homodimers on the MORE (E) or Sox2-Oct heterodimers on the SoxOct element (F) are shown for a panel of six POU proteins. The mean is shown with standard deviation as error bars (n≥3) and Tukey multiple comparison of means was used for assessment of statistical significances (*** p < 0.001, ** p < 0.01, * p < 0.05). N.A.: not assessed.


G . Properties of Oct4 Oct6 homodimers from molecular dynamics ( MD ) simulations . structural snapshots illustrate Oct6 ( first monomer in orange , second in blue ) 3 parameters used : drmsd = root mean square deviation of interatomic distances measured for backbone of structured domains ( excluding linker ) ; θ = angle describing kink in helix α4 ( data for both monomers 1 2 are shown ) ; d = between centers of mass of 2 helices α5 ; w = minor groove width in region between 2 monomers .

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G. Properties of Oct4 and Oct6 homodimers from molecular dynamics (MD) simulations. The structural snapshots illustrate Oct6 (first monomer in orange, second in dark blue) and the 3 parameters used: drmsd = the root mean square deviation of interatomic distances measured for the backbone of the structured domains (excluding the linker); θ = the angle describing the kink in helix α4 (data for both monomers 1 and 2 are shown); d = the distance between the centers of mass of the 2 helices α5; w = the DNA minor groove width in the region between the 2 monomers.


A . Zoom - in view of Oct6 - Oct6 homodimer interface in molecular models on crystal structures of two POU homodimers on MORE . Oct6 POUS , orange ; POUHD of other monomer , blue . Important residues are labelled Met ( Oct6 ) in position 151 which was identified to guide - binding preference is marked with a star . helixes of POU domains are numbered . Colour representation as in Fig . 1B , 1C . For clarity , second monomer of Oct4 / Oct6 is shown in blue .

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A. Zoom-in view of the Oct6-Oct6 homodimer interface in the molecular models based on crystal structures of two POU homodimers on MORE DNA. Oct6 POUS, orange; POUHD of the other monomer, blue. Important residues are labelled and Met (Oct6) in position 151 which was identified to guide the DNA-binding preference is marked with a star. Individual helixes of POU domains are numbered. Colour representation as in Fig. 1B, 1C. For clarity, the second monomer of Oct4/Oct6 is shown in dark blue.


B . C . Network of hydrophobic interactions in Oct6 ( B ) Oct4 ( C ) . numbers show minimal interatomic distances ( excluding hydrogens ) between residues involved calculated from simulations . In blue are those distances that change significantly between Oct4 Oct6 in red those unchanged . shown standard deviations represent a measure for variability during simulations not for uncertainty in calculations .

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B. and C. Network of hydrophobic interactions in Oct6 (B) and Oct4 (C). The numbers show the minimal interatomic distances (excluding hydrogens) between the residues involved calculated from the simulations. In blue are those distances that change significantly between Oct4 and Oct6 and in red those unchanged. The shown standard deviations represent a measure for the variability during the simulations and not for the uncertainty in the calculations.


D . EMSA showing a differential binding of WT Oct4 Oct6 POU domains as well as mutated Oct4151M Oct6151S POU domains on MORE ( OctOct ) element . free bound by monomer or dimer are indicated .

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D. EMSA showing a differential binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains on MORE (OctOct) element. The free DNA and DNA bound by monomer or dimer are indicated.


E . EMSA showing binding of WT Oct4 Oct6 POU domains as well as mutated Oct4151M Oct6151S POU domains to SoxOct element in absence or presence of Sox2 HMG . free bound by monomers or dimer are indicated . Quantifications of cooperativity measurements are shown in Fig . EV3C .

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E. EMSA showing binding of WT Oct4 and Oct6 POU domains as well as mutated Oct4151M and Oct6151S POU domains to SoxOct element in the absence or presence of the Sox2 HMG. The free DNA and DNA bound by monomers or dimer are indicated. Quantifications of cooperativity measurements are shown in Fig. EV3C.


F . Bar plot on single tube EMSAs using cy5 - SoxOct FAM - MORE elements showing difference in - binding of WT Oct4 , WT Oct6 , Oct4151M Oct6151S POU domains . mean log2 of cy5 ( heterodimer ) FAM ( homodimer ) band intensities are depicted with standard deviation ( n = 9 ) . data points are shown as gray jitter plots . Tukey multiple comparisons of means was performed to assess significance ( * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , p > 0 . 05 n . s . ) . Compare to Fig . EV1D EV3D .

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F. Bar plot based on single tube EMSAs using cy5-SoxOct and FAM-MORE DNA elements showing the difference in DNA-binding of WT Oct4, WT Oct6, Oct4151M and Oct6151S POU domains. The mean log2 ratio of cy5 (heterodimer) and FAM (homodimer) band intensities are depicted with standard deviation (n=9). Individual data points are shown as gray jitter plots. Tukey multiple comparisons of means was performed to assess significance (*** p < 0.001, ** p < 0.01, * p < 0.05, p > 0.05 n.s.). Compare to Fig. EV1D and EV3D.


A B . Molecular models show position of mutated residues in structures of two dimers on : Sox2 - Oct4 heterodimer ( A ) Oct4 - Oct4 homodimer ( B ) . Ser in position 151 ( asterisk ) was mutated to Met in order to shift preference of Oct4 for homodimerization . helixes of POU domains are numbered .

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A and B. Molecular models show the position of mutated residues in the structures of two dimers on DNA: Sox2-Oct4 heterodimer (A) and Oct4-Oct4 homodimer (B). Ser in position 151 (asterisk) was mutated to Met in order to shift the preference of Oct4 for homodimerization. The individual helixes of the POU domains are numbered.


D . GFP - positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2 , Klf4 , c - Myc . Colonies were imaged 16 days after second viral infection , using a microscope . Scale bars : 250 μm ; objective 10x .

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D. GFP-positive colonies of mouse iPSCs generated by Oct4 mutants in combination with Sox2, Klf4, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.


B . Oct4 - GFP - positive colonies of two O6SKM iPSC lines , expanded from single colonies of pluripotent that were generated by contribution of synthetic Oct6 molecule ( Oct67K , 22T , LinkO4 , 151S ) . Scale bars : 250 μm .

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B. Oct4-GFP-positive colonies of two O6SKM iPSC lines, expanded from single colonies of pluripotent cells that were generated by the contribution of the synthetic Oct6 molecule (Oct67K,22T,LinkO4,151S). Scale bars: 250 μm.


C . Genotyping of two O6SKM iPSC lines . Two stable lines of iPSCs generated with Oct6 , Sox2 , Klf4 , c - Myc retroviral set are positive for these four transgenes but negative for Oct4 transgene . OSKM iPSCs OG2 - MEFs were used as PCR controls .

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C. Genotyping of two O6SKM iPSC lines. Two stable lines of iPSCs generated with the Oct6, Sox2, Klf4, and c-Myc retroviral set are positive for these four transgenes but negative for the Oct4 transgene. OSKM iPSCs and OG2-MEFs were used as PCR controls.


D . Expression of viral transgenes in two O6SKM iPSC lines done by qRT - PCR using specific primers . OG2 - MEFs were harvested 5 days after infection used for comparison as a positive control .

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D. Expression analysis of viral transgenes in two O6SKM iPSC lines done by qRT-PCR using specific primers. OG2-MEFs were harvested 5 days after infection and used for comparison as a positive control.


E . Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 Nanog . Oct4 expression was confirmed by Oct4 - GFP transgene . stained by DAPI . Scale bars : 100 μm .

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E. Two O6SKM iPSC lines immunostained for pluripotency markers Sox2 and Nanog. Oct4 expression was confirmed by the Oct4-GFP transgene. DNA stained by DAPI. Scale bars: 100 μm.


F . Bisulfite sequencing of genomic Oct4 , Nanog , Col1a1 promoter regions of O6SKM iPSCs , OSKM iPSCs , OG2 - MEFs . black circles represent unmethylated methylated CpG sites , respectively .

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F. Bisulfite sequencing of genomic Oct4, Nanog, and Col1a1 promoter regions of O6SKM iPSCs, OSKM iPSCs, and OG2-MEFs. White and black circles represent unmethylated and methylated CpG sites, respectively.


G . Pairwise scatter plots comparing global gene expression profile of O6SKM iPSCs1 with OG2 ESCs ( left ) O4SK iPSCs ( right ) . Black lines represent a two - fold change in gene expression between paired lines . On right side of plots , color bar indicates scattering density . Red green dots represent up - downregulated genes , respectively . Positions of selected pluripotency - related genes are highlighted as orange points .

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G. Pairwise scatter plots comparing the global gene expression profile of O6SKM iPSCs1 with OG2 ESCs (left) and O4SK iPSCs (right). Black lines represent a two-fold change in gene expression levels between the paired cell lines. On the right side of the plots, the color bar indicates scattering density. Red and green dots represent up- and downregulated genes, respectively. Positions of selected pluripotency-related genes are highlighted as orange points.


H . Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs O4SK iPSCs , illustrating close relationship between their global gene expression profiles .

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H. Hierarchical clustering of O6SKM iPSC lines with OG2 ESCs and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.


A . In vitro differentiation of O6SKM iPSC lines into of three germ layers as shown by immunochemistry : endoderm ( α - fetoprotein , AFP ) , mesoderm ( α - smooth muscle actin , SMA ) , ectoderm ( α - tubulin , TUJ1 ) . Nuclei ( ) were stained by Hoechst ( blue ) . Scale bars : 200 μm .

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A. In vitro differentiation of O6SKM iPSC lines into cells of all three germ layers as shown by immunochemistry: endoderm (α-fetoprotein, AFP), mesoderm (α-smooth muscle actin, SMA), and ectoderm (α-tubulin, TUJ1). Nuclei (DNA) were stained by Hoechst (blue). Scale bars: 200 μm.


B . F1 offspring of chimera mice with contribution from O6SKM iPSCs CD1 mice .

B. F1 offspring of chimera male mice with contribution from O6SKM iPSCs and CD1 female mice.


C . Pictures demonstrating germline transmission ; endogenous Oct4 - GFP signal was detected in gonads of F1 pups . Scale bars : 200 μm .

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C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.


( A ) Top panel displays structure of each CDK4 / 6 inhibitor tested . Bottom panel shows dose response curves with these inhibitors displaying of G1 - arrested RPE1 - FUCCI . To obtain dose response curves , number of mKO - Cdt1 positive ( G1 - arrested ) were calculated following 24hr drug addition ( blue solid lines ) or 24h after subsequent drug washout ( blue dotted lines ) . Cmax values observed in ( taken from ( He et al . , 2017 ; Klein et al . , 2018 ) ) are represented on each graph with red dotted lines . Graphs display mean data - / + from 3 experiments , with at least 500 counted per condition per experiment . ( B ) of G1 - arrested RPE1 - FUCCI , calculated as in panel A , but using a fixed of CDK4 / 6 inhibitor for different durations of time , as indicated . Note , this is a fixed that quantifies of G1 - arrested prior to , or 24 hrs following , CDK4 / 6 inhibitor washout . Each bar displays mean data + from 3 experiments , with at least 500 counted per condition per experiment . (

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(A) Top panel displays structure of each CDK4/6 inhibitor tested. Bottom panel shows dose response curves with these inhibitors displaying percentage of G1-arrested RPE1-FUCCI cells. To obtain dose response curves, the number of mKO-Cdt1 positive (G1-arrested) cells were calculated following 24hr drug addition (dark blue solid lines) or 24h after subsequent drug washout (light blue dotted lines). Cmax values observed in patients (taken from (He et al., 2017; Klein et al., 2018)) are represented on each graph with red dotted lines. Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (B) Percentage of G1-arrested RPE1-FUCCI cells, calculated as in panel A, but using a fixed concentration of CDK4/6 inhibitor for different durations of time, as indicated. Note, this is a fixed assay that quantifies the percentage of G1-arrested cells prior to, or 24 hrs following, CDK4/6 inhibitor washout. Each bar displays mean data + SEM from 3 experiments, with at least 500 cells counted per condition per experiment. (


( A ) Dose response curves displaying of G1 - arrested p53 - WT ( blue ) or KO ( green ) RPE1 - FUCCI following 24hr incubation with palbociclib ( solid lines ) or 24h after subsequent washout ( dotted lines ) . Graphs display mean data - / + from 3 experiments , with at least 500 counted per condition per experiment .

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(A) Dose response curves displaying the percentage of G1-arrested p53-WT (blue) or KO (green) RPE1-FUCCI cells following 24hr incubation with palbociclib (dark solid lines) or 24h after subsequent washout (light dotted lines). Graphs display mean data -/+ SEM from 3 experiments, with at least 500 cells counted per condition per experiment.


( C ) Representative quantifications of colony forming assays in p53 - WT or KO RPE1 treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days then grown at low density without inhibitor for 10 days . Each bar displays mean data + from 3 experiments .

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(C) Representative images and quantifications of colony forming assays in p53-WT or KO RPE1 cells treated with palbociclib (1.25μM) for 1, 4 or 7 days and then grown at low density without inhibitor for 10 days. Each bar displays mean data + SEM from 3 experiments.


( E ) of cycle profiles from treated as in D . Graph shows mean of + SD that divide at each round of division , with 150 analysed in total from 3 experimental repeats .

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(E) Quantification of cell cycle profiles from cells treated as in D. Graph shows the mean percentage of cells + SD that divide at each round of division, with 150 cells analysed in total from 3 experimental repeats.


( A ) Representative immunofluorescence of p21 in p53 - WT or KO RPE1 , 48 hours after release from 1 , 4 or 7 days palbociclib ( 1 . 25μM ) treatment . Zoom inserts are 3x magnification of indicated regions . Scale bars = 250 μM . ( B ) of p21 intensities in treated as in panel A . At least 100 were analysed per experiment graph shows data from 3 experimental repeats . Violin plots display variation in intensities between . Horizontal lines display median , error bars show 95 % confidence intervals . (

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(A) Representative immunofluorescence images of p21 levels in p53-WT or KO RPE1 cells, 48 hours after release from 1, 4 or 7 days palbociclib (1.25μM) treatment. Zoom inserts are 3x magnification of the indicated regions. Scale bars = 250 μM. (B) Quantification of p21 intensities in cells treated as in panel A. At least 100 cells were analysed per experiment and graph shows data from 3 experimental repeats. Violin plots display the variation in intensities between individual cells. Horizontal lines display the median, and error bars show 95% confidence intervals. (


( D - E ) of morphologies ( D ) γH2AX - positive damage foci ( E ) following palbociclib ( 1 . 25μM ) treatment in p53 WT KO RPE1 . were treated for 1 , 4 or 7 days then analysed before or after drug washout for 48 hours . 100 ( ) or 50 ( γH2AX foci ) were scored per condition per experiment , bar graphs represent mean data + from 6 experiments .

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(D-E) Quantification of nuclear morphologies (D) and γH2AX-positive DNA damage foci (E) following palbociclib (1.25μM) treatment in p53 WT and KO RPE1 cells. Cells were treated for 1, 4 or 7 days and then analysed before or after drug washout for 48 hours. 100 cells (nuclear morphology) or 50 cells (γH2AX foci) were scored per condition per experiment, and bar graphs represent mean data +SEM from 6 experiments.


( J - K ) Representative immunofluorescence ( J ) quantifications ( K ) of mitotic replication assays ( MiDAS ) in p53 - KO RPE1 released from 7 days of palbociclib ( 1 . 25μM ) treatment or following 0 . 4uM aphidicolin treatment for 40Hrs . EdU foci were quantified in nocodazole - arrested . Scale bar = 5 μM , zoom inserts = 3x magnification of highlighted areas . 10 were analysed per experiment bar chart shows mean + from 3 experimental repeats .

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(J-K) Representative immunofluorescence images (J) and quantifications (K) of mitotic DNA replication assays (MiDAS) in p53-KO RPE1 cells released from 7 days of palbociclib (1.25μM) treatment or following 0.4uM aphidicolin treatment for 40Hrs. EdU foci were quantified in nocodazole-arrested cells. Scale bar= 5 μM, zoom inserts = 3x magnification of highlighted areas. 10 cells were analysed per experiment and the bar chart shows the mean + SEM from 3 experimental repeats.


( A ) Volcano plot of up or downregulated following prolonged palbociclib ( 1 . 25μM ) treatment in RPE1 . top 10 significantly upregulated downregulated are shown in blue red , respectively .

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(A) Volcano plot of proteins up or downregulated following prolonged palbociclib (1.25μM) treatment in RPE1 cells. The top 10 significantly upregulated and downregulated proteins are shown in blue and red, respectively.


( D ) Representative western blots of whole lysates from RPE1 - WT treated with palbociclib ( 1 . 25μM ) for 1 , 4 or 7 days , or treated identically , then washed out for indicated times to reflect when majority of are in S - phase ( see Figure 1C ) .

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(D) Representative western blots of whole cell lysates from RPE1-WT cells treated with palbociclib (1.25μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times to reflect when the majority of cells are in S-phase (see Figure 1C).


( F - G ) Representative ( F ) quantifications ( G ) of colony forming assays with RPE1 treated with palbociclib ( 1 . 25μM ) for indicated times , then grown at low density without palbociclib for 10 days . DMSO ( control ) or indicated genotoxic drugs were applied for first 24 h after palbociclib washout . Each bar displays mean data + SD from 4 experiments .

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(F-G) Representative images (F) and quantifications (G) of colony forming assays with RPE1 cells treated with palbociclib (1.25μM) for indicated times, and then grown at low density without palbociclib for 10 days. DMSO (control) or indicated genotoxic drugs were applied for the first 24 h after palbociclib washout. Each bar displays mean data + SD from 4 experiments.


( A ) Representative western blots of whole lysates from MCF7 treated with palbociclib ( 1 μM ) for 1 , 4 or 7 days , or treated identically , then washed out for indicated times ( see Figure 1C ) . ( B ) of adjusted relative density from 3 independent western blot experiments . Bars display mean values - / + SD . Significance determined by unpaired Student ' s T comparing treated target to asynchronous target control . ( * < 0 . 01 , * * < 0 . 001 , * * * < 0 . 0001 ) .

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(A) Representative western blots of whole cell lysates from MCF7 cells treated with palbociclib (1 μM) for 1, 4 or 7 days, or treated identically, and then washed out for the indicated times (see Figure 1C). (B) Analysis of adjusted relative density from 3 independent western blot experiments. Bars display mean values -/+ SD. Significance determined by unpaired Student's T test comparing treated target protein to asynchronous target control. (* < 0.01, ** < 0.001, *** < 0.0001).


( C , D ) of morphologies ( C ) or γH2AX - positive damage foci ( D ) from MCF7 , MCF7 p53 KO or T47D that were treated with palbociclib ( 1μM ) for 0 , 1 , 4 , or 7 days then analysed 48hr after drug washout . Either 100 ( ) or 50 ( γH2AX ) were scored per condition per experiment bar graphs represent mean data + from 3 experiments .

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(C,D) Quantification of the nuclear morphologies (C) or γH2AX-positive DNA damage foci (D) from MCF7, MCF7 p53 KO or T47D cells that were treated with palbociclib (1μM) for 0, 1, 4, or 7 days and then analysed 48hr after drug washout. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( F ) of colony forming assays of MCF7 , MCF7 p53 KO T47D treated with CDK4 / 6 inhibitor for 0 , 1 , 4 or 7 days then grown at a low density without palbociclib for 14 - 21 days . Bar graphs displays mean data + from 4 - 5 experiments .

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(F) Quantification of colony forming assays of MCF7, MCF7 p53 KO and T47D cells treated with CDK4/6 inhibitor for 0, 1, 4 or 7 days and then grown at a low density without palbociclib for 14-21 days. Bar graphs displays mean data + SEM from 4-5 experiments.


( B , C ) of morphologies ( B ) or γH2AX - positive damage foci ( C ) from different cancer lines , as indicated , treated continuously with palbociclib ( 1μM ) for 0 , 1 , 2 , or 3 . Either 100 ( ) or 50 ( γH2AX ) were scored per condition per experiment bar graphs represent mean data + from 3 experiments .

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(B,C) Quantification of the nuclear morphologies (B) or γH2AX-positive DNA damage foci (C) from different cancer lines, as indicated, treated continuously with palbociclib (1μM) for 0, 1, 2, or 3 weeks. Either 100 cells (nuclear morphology) or 50 cells (γH2AX) were scored per condition per experiment and bar graphs represent mean data + SEM from 3 experiments.


( D ) of weekly proliferation rate in treated as in B . A total of 60 , 000 of each were cultured in 10cm dishes with 1uM palbociclib for a total of 3 . Every 7 days , were trypsinised total were determined . 0 - week timepoint shows 7 - day fold increase of untreated . data points show fold increase in over each 7 - day period , bars represent mean from 2 experiments .

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(D) Quantification of weekly proliferation rate in cells treated as in B. A total of 60,000 cells of each cell line were cultured in 10cm dishes with 1uM palbociclib for a total of 3 weeks. Every 7 days, cells were trypsinised and total cell counts were determined. The 0-week timepoint shows the 7-day fold increase of untreated cells. The data points show fold increase in cell count over each 7-day period, and bars represent the mean from 2 experiments.


( A ) RPE1 were transfected with either scramble or TMEM135 siRNAs immunostained for LAMP1 as a lysosome marker ( red ) PMP70 as a peroxisome marker ( green ) . Scale bar , 10 μm . ( B ) of overlap signal between lysosomes peroxisomes shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 50 were scored per condition per experiment . * P < 0 . 05 , Student ' s t - .

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(A) RPE1 cells were transfected with either scramble or TMEM135 siRNAs and immunostained for LAMP1 as a lysosome marker (red) and PMP70 as a peroxisome marker (green). Scale bar, 10 μm. (B) Quantification of overlap signal between lysosomes and peroxisomes shown in (A). Data represent mean ± SD (n=3 experiments), 50 cells were scored per condition per experiment. *P < 0.05, Student's t-test.


( C ) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom , separated by SDS - PAGE , immunoblotted for LAMP1 , EEA1 , Rab11 , Rab8 , PMP70 , GM130 , Na + / K + ATPase .

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(C) Subcellular fractions of a discontinuous sucrose gradient collected from top to bottom, separated by SDS-PAGE, and immunoblotted for LAMP1, EEA1, Rab11, Rab8, PMP70, GM130, and Na+/K+ ATPase.


( D ) of total cholesterol in subcellular fractions Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - .

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(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( E ) transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage for precursor of SREBP2 SREBP2 .

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(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.


( F ) transfected with TMEM135 siRNA were subjected to qPCR . Data represent mean ± SD ( n = 3 experiments ) , * P < 0 . 05 , Student ' s t - .

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(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNAs as indicated , followed by serum starvation for 24 h , immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( B ) of of ciliated shown in ( A ) . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by serum starvation for 24 h, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (B) Quantification of the percentage of ciliated cells shown in (A). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) were transfected with siRNAs as indicated incubated with HPβCD in serum - starvation media followed by filipin . Scale bar , 20 μm .

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(C) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.


( D ) were transfected with siRNAs shown in ( C ) immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( E ) of of ciliated shown in ( D ) . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(D) Cells were transfected with siRNAs shown in (C) and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (E) Quantification of the percentage of ciliated cells shown in (D). Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( F ) were transfected with siRNAs as indicated , incubated with cholesterol - MβCD in serum - starvation media , immunostained for ARL13B ( red ) γ - tubulin ( green ) . Scale bar , 10 μm . ( G ) of of ciliated shown in ( F ) . Data represent mean ± SD ( n = 3experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(F) Cells were transfected with siRNAs as indicated, incubated with cholesterol-MβCD complex in serum-starvation media, and immunostained for ARL13B (red) and γ-tubulin (green). Scale bar, 10 μm. (G) Quantification of the percentage of ciliated cells shown in (F). Data represent mean ± SD (n=3experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNA as indicated , immunostained for Rab8 ( red ) γ - tubulin ( green ) in either 10 % FBS medium ( upper panel ) or serum - starvation medium ( lower panel ) . Scale bar , 10 μm . Arrowhead indicates Rab8 localized to centrioles .

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(A) RPE1 cells were transfected with siRNA as indicated, and immunostained for Rab8 (red) and γ-tubulin (green) in either 10% FBS medium (upper panel) or serum-starvation medium (lower panel). Scale bar, 10 μm. Arrowhead indicates Rab8 localized to the centrioles.


( B ) of of with Rab8 localized to centriole Data represent mean ± SD ( n = 3 experiments ) , 200 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(B) Quantification of the percentage of cells with Rab8 localized to the centriole Data represent mean ± SD (n=3 experiments), 200 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) were transfected with siRNA immunostained for IFT20 ( red ) γ - tubulin ( green ) . Scale bar , 10 μm .

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(C) Cells were transfected with siRNA and immunostained for IFT20 (red) and γ-tubulin (green). Scale bar, 10 μm.


( D ) of of IFT20 at centrioles Data represent mean ± SD ( n = 3 experiments ) , 150 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(D) Quantification of the percentage of IFT20 intensity at the centrioles Data represent mean ± SD (n=3 experiments), 150 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) RPE1 were transfected with siRNAs as indicated , followed by transfection with wild - type pGFP - Rab8a ( WT - Rab8 ) , constitutively active pGFP - Rab8a ( Q67L ) ( CA - Rab8 ) , or DN Rab8 dominant - negative pGFP Rab8a ( T22N ) , incubated in serum - starved media for 12 h , immunostained for ARL13B ( Red ) , GFP - Rab8 ( green ) , DAPI ( blue ) . Scale bar , 10 μm .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by transfection with wild-type pGFP-Rab8a (WT-Rab8), constitutively active pGFP-Rab8a (Q67L) (CA-Rab8), or DN Rab8 dominant-negative pGFP Rab8a (T22N), incubated in serum-starved media for 12 h, and immunostained for ARL13B (Red), GFP-Rab8 (green), and DAPI (blue). Scale bar, 10 μm.


( B ) of of ciliated Data represent mean ± SD ( n = 3 experiments ) , 200 GFP - positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(B) Quantification of the percentage of ciliated cells Data represent mean ± SD (n=3 experiments), 200 GFP-positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( C ) ( Upper panel ) were transfected as indicated lysates were incubated with purified , including GST or GST - JCF1 ( RBD ) . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by western blot with Rab8 antibody . ( Lower panel ) of bands was quantified by Image J software . amount of GTP - Rab8 were normalized to control level . Bar graph represents mean ± SD ( n = 3 experiments ) . * P < 0 . 05 , Student ' s t - .

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(C) (Upper panel) Cells were transfected as indicated and the cell lysates were incubated with purified proteins, including GST or GST-JCF1 (RBD). The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by western blot with Rab8 antibody. (Lower panel) Intensity of the bands was quantified by Image J software. The amount of GTP-Rab8 were normalized to the control level. Bar graph represents mean ± SD (n=3 experiments). *P < 0.05, Student's t-test.


( D ) were transfected lysates were incubated with purified GST - JCF1 ( RBD ) fusion . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody . ( Lower panel ) were transfected lysates were incubated with purified GST - . amount of GTP - Rab8 bound to GFT - JCF1 ( RBD ) was analyzed by Western blot with Rab8 antibody .

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(D) Cells were transfected and cell lysates were incubated with purified GST-JCF1 (RBD) fusion protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody. (Lower panel) Cells were transfected and cell lysates were incubated with purified GST- protein. The amount of GTP-Rab8 bound to GFT-JCF1(RBD) was analyzed by Western blot with Rab8 antibody.


( A ) RPE1 were transfected with siRNAs as indicated , followed by 24 h incubation in serum - starved media . were then subjected to fractionation , Western blot for IFT20 , Golgi marker GM130 , marker UBXD8 , marker CREB .

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(A) RPE1 cells were transfected with siRNAs as indicated, followed by 24 h incubation in serum-starved media. Cells were then and subjected to fractionation, and Western blot for IFT20, the Golgi marker GM130, membrane marker UBXD8, and nuclear marker CREB.


( B ) Efficiency of IFT20 knockdown by western blot .

(B) Efficiency of IFT20 knockdown by western blot.


( C ) were transfected with siRNAs as indicated , followed by incubation in serum - starved media for 24 h , immunostained for ARL13B ( red ) . Scale bar , 10 μm . bar graph represents of of ciliated . Data represent mean ± SD ( n = 3 experiments ) , 250 were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(C) Cells were transfected with siRNAs as indicated, followed by incubation in serum-starved media for 24 h, and immunostained for ARL13B (red). Scale bar, 10 μm. The bar graph represents the quantification of the percentage of ciliated cells. Data represent mean ± SD (n=3 experiments), 250 cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( D ) were transfected immunostained for Rab8 γ - tubulin , followed by of of with Rab8 localized to centriole . Data represent average ( n = 2 experiments ) .

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(D) Cells were transfected and immunostained for Rab8 and γ-tubulin, followed by quantification of the percentage of cells with Rab8 localized to the centriole. Data represent average (n=2 experiments).


( E ) were transfected with siRNAs as indicated , followed by transfection with Flag - IFT20 , incubation in serum - starvation media for 12 h , immunostained for ARL13B . Representative of Flag - IFT20 ( green ) , ARL13B ( red ) , DAPI ( blue ) are shown . Scale bar , 10 μm . ( F ) of of ciliated with both Flag - IFT20 ARL13B localized in cilium . Data represent mean ± SD ( n = 3 experiments ) , 150 Flag positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(E) Cells were transfected with siRNAs as indicated, followed by transfection with Flag-IFT20, incubation in serum-starvation media for 12 h, and immunostained for ARL13B. Representative fluorescent images of Flag-IFT20 (green), ARL13B (red), and DAPI (blue) are shown. Scale bar, 10 μm. (F) Quantification of the percentage of ciliated cells with both the Flag-IFT20 and ARL13B localized in the cilium. Data represent mean ± SD (n=3 experiments), 150 Flag positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( G ) were transfected with siRNAs as indicated , followed by further transfection with CA - Rab8 , incubation in serum - starvation media for 12 h , immunostained for acetylated - tubulin . Representative of GFP - Rab8 Q67L ( green ) , acetylated - tubulin ( red ) , ( blue ) are shown . Scale bar , 10 μm . ( H ) of of GFP - positive ciliated ( only those cilia having both GFP - Rab8 acetylated - tubulin on cilium were considered for ) . Data represent mean ± SD ( n = 3 experiments ) , 200 GFP positive were scored per condition per experiment , * P < 0 . 05 , Student ' s t - .

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(G) Cells were transfected with siRNAs as indicated, followed by further transfection with CA-Rab8, incubation in serum-starvation media for 12 h, and immunostained for acetylated-tubulin. Representative fluorescent images of GFP-Rab8 Q67L (green), acetylated-tubulin (red), and DNA (blue) are shown. Scale bar, 10 μm. (H) Quantification of the percentage of GFP -positive ciliated cells (only those cilia having both GFP-Rab8 and acetylated-tubulin on cilium were considered for quantification). Data represent mean ± SD (n = 3 experiments), 200 GFP positive cells were scored per condition per experiment, *P < 0.05, Student's t-test.


( A ) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 .

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(A) Efficiency of Rab8a depletion confirmed by Western blot in RPE1 cells.