Western blot detection of presence of His - STING in immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS His - STING were incubated together in IP lysis buffer overnight at 4 ° C .

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Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C.


Western blot detection of presence of GST - cGAS His - STING in immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS His - STING were incubated together in absence or presence of increasing 2 ′ 3 ′ - cGAMP .

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Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP.


Western blot detection of presence of cGAS STING in immunoprecipitates of streptavidin beads . THP - 1 were stimulated with biotin - ecGAMP ( 5 μg / ml ) or biotin - icGAMP ( 1 μg / ml ) for indicated time lysates were harvested for IP with streptavidin beads . Data ( A - B , D - G ) are representative of 3 independent experiments .

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Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.


qRT - PCR detection of Ifnb1 mRNA ( A ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc was used for statistical . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , p < 0 . 001 .

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qRT-PCR detection of Ifnb1 mRNA (A) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


ELISA detection of IFNβ in supernatants ( B ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc was used for statistical . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , p < 0 . 001 .

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ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


Western blot detection of indicated in lysates of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are representative of 3 independent experiments .

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Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.


( A ) WT atg1Δ grown in YEPD medium were transferred to SD - N or SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours , dead stained by phloxine B were observed by microscopy . Scale bar , 25 µm .

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(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm.


( B - C ) WT atg1Δ grown nitrogen starved as ( A ) for indicated times . viability ( B ) medium pH ( C ) were examined by phloxine B pH meter , respectively . These data represent average of three independent experiments bars indicate standard deviations .

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(B-C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT atg1Δ grown in YEPD medium were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) . After 120 hours , cultures were diluted 5 . 0 × 105 fold plated onto YEPD agar . plates were incubated at 30 ° C for four days . Insets indicate plate overlaid with TTC agar to examine respiratory competency of formed colonies .

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(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N +50 mM MES-KOH (pH 6.2). After 120 hours, cultures were diluted 5.0×105 fold and plated onto YEPD agar. The plates were incubated at 30°C for four days. Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies.


( B ) WT , atg1Δ were nitrogen - starved as ( A ) . viability respiratory competency was determined by phloxine B TTC overlay technique as ( A ) , respectively . black gray areas indicate of viable that are respiratory competent or respiratory deficient , respectively .

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(B) WT, and atg1Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( C ) WT , atg1Δ , atg2Δ , atg7Δ , atg11Δ , atg15Δ , atg32Δ were nitrogen - starved as ( A ) . viability respiratory competency was determined by phloxine B TTC overlay technique as ( A ) , respectively . black gray areas indicate of viable that are respiratory competent or respiratory deficient , respectively . These data represent average of three independent experiments bars indicate standard deviations .

(C) WT, atg1Δ, atg2Δ, atg7Δ, atg11Δ, atg15Δ, and atg32Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) WT atg1Δ expressing mitochondria targeted mCherry cultured in SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for 120 hours were observed by microscopy . Mitochondrial was stained with SYBR green I , mitochondria were visualized by mitochondria targeted mCherry . Scale bar , 2 µm .

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(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm.


( B - C ) WT atg1Δ were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for indicated time . ROS accumulation was detected by DHE ( B ) . Each photo contains about 200 . Scale bar , 20 µm . ( C ) shows of ROS accumulated ( n > 200 ) .

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(B-C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells).


( D ) WT atg1Δ were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) with 10 mM NAC for indicated time . viability was determined by phloxine B . from these cultures were plated on YEPD agar overlaid with TTC agar to examine respiratory competency . black gray areas indicate of viable that are respiratory competent or respiratory deficient , respectively . These data represent average of three independent experiments bars indicate standard

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(D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations


( A ) WT atg1Δ were transferred to SD - N + 50 mM MES - KOH ( pH 6 . 2 ) medium for indicated time . Lysates were prepared using alkaline - trichloroacetic acid method subjected to immunoprecipitation with anti - Cox2 , anti - Cox4 , anti - Tim17 anti - Pgk1 . Pgk1 was used as loading control .

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(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control.


( B ) WT atg1Δ expressing Cta1 - 3 × FLAG or Ctt1 - 3 × FLAG were nitrogen - starved as in ( A ) for indicated time . lysates were prepared as ( A ) subjected to immune precipitation with anti - FLAG anti - Pgk1 . Pgk1 was used as loading control .

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(B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.


( A ) WT atg1Δ were transferred to non - buffered SD - N medium for indicated time . viability was determined by phloxine B . Nitrogen - starved were plated onto YEPD agar overlaid with TTC agar to examine respiratory competency of formed colonies . black gray areas indicate of viable that are respiratory competent or respiratory deficient , respectively .

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(A) WT and atg1Δ cells were transferred to non-buffered SD-N medium for the indicated time. Cell viability was determined by phloxine B staining. Nitrogen-starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively.


( B ) WT , atg1Δ , rho0 , rho0 atg1Δ grown in YEPD medium were transferred to SD - N with or without 10 mM NAC for indicated time . In presence of NAC , medium pH was adjusted by using KOH . viability medium pH were examined by phloxine B pH meter , respectively . These data represent average of three independent experiments bars indicate standard deviations .

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(B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.


( A ) Quality of fractionation assays was controlled by western blot by assessing distribution of markers in whole extract ( WCE ) , cytosol nucleus . lysates were isolated from mouse neocortical ( 14 days in culture ) treated for 3hrs with transcription inhibitor triptolide ( 1µM ) . ( four independent cultures )

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(A) Quality of the cell fractionation assays was controlled by western blot by assessing the distribution of nuclear and cytosolic markers in the whole cell extract (WCE), the cytosol and the nucleus. Protein lysates were isolated from mouse primary neocortical cells (14 days in culture) treated for 3hrs with the transcription inhibitor triptolide (1µM). (four independent cultures)


Efficiencies of bicuculline ( A ) stimulation were assessed by controlling induction of ERK phosphorylation by immunostaining . Mouse neocortical ( 14 days in culture ) were stimulated with bicuculline ( 20µM ) to elevate neuronal network excitation were stained with anti - phospho - ERK ( green , bottom ) anti - MAP2 ( , top ) antibodies counterstained with Hoechst ( blue , top ) . ( four independent cultures )

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Efficiencies of bicuculline (A) stimulation were assessed by controlling the induction of ERK phosphorylation by immunostaining. Mouse primary neocortical cells (14 days in culture) were stimulated with bicuculline (20µM) to elevate the neuronal network excitation Cells were stained with anti-phospho-ERK (green, bottom) and anti-MAP2 (white, top) antibodies and counterstained with Hoechst (blue, top). (four independent cultures)


RT - PCR validations of regulated IR - transcripts through splicing upon bicuculline stimulation ( C ) Expression of IR - isoforms spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means SDs ( standard deviations ) of PIR values are shown beneath each panel . In addition , fold changes in expression of IR - transcripts ( red orange ) spliced ( grey ) isoforms were assessed with real - time qPCR using three different primer sets , as represented in top scheme . Note that Gria3 spliced transcript ( C ) does not correspond to canonical mRNAs presumably arise from a first step of recursive splicing thereby likely require splicing completion to generate fully mature Gria3 transcripts ( Sibley et al , 2015 ) .

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RT-PCR validations of regulated IR-transcripts through splicing upon bicuculline stimulation (C) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme. Note that the Gria3 spliced transcript (C) does not correspond to the canonical mRNAs and presumably arise from a first step of recursive splicing and thereby likely require splicing completion to generate fully mature Gria3 transcripts (Sibley et al, 2015).


RT - PCR validations of regulated IR - transcripts through splicing upon BDNF stimulation ( D ) Expression of IR - isoforms spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means SDs ( standard deviations ) of PIR values are shown beneath each panel . In addition , fold changes in expression of IR - transcripts ( red orange ) spliced ( grey ) isoforms were assessed with real - time qPCR using three different primer sets , as represented in top scheme .

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RT-PCR validations of regulated IR-transcripts through splicing upon BDNF stimulation (D) Expression of the IR-isoforms and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs (standard deviations) of PIR values are shown beneath each panel. In addition, fold changes in the expression of the IR-transcripts (red and orange) and spliced (dark grey) isoforms were assessed with real-time qPCR using three different primer sets, as represented in the top scheme.


( A ) of IR - transcript expression values in control , bicuculline - stimulated BDNF - stimulated conditions . IR - transcripts regulated in at least one condition are displayed ( fold change of intron - retaining transcript expression ≥ 20 % | z - score | ≥ 1 . 5 ) .

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(A) Heatmap of the IR-transcript expression values in control, bicuculline-stimulated and BDNF-stimulated conditions. IR-transcripts regulated in at least one condition are displayed (fold change of intron-retaining transcript expression ≥ 20% and |z-score| ≥ 1.5).


RT - PCR validations of IR - transcripts commonly regulated ( C ) Expression of IR - transcripts spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in expression of IR - transcripts ( red orange ) spliced isoforms ( grey ) were assessed with real - time qPCR .

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RT-PCR validations of IR-transcripts commonly regulated (C) Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.


RT - PCR validations of IR - transcripts , specifically regulated upon bicuculline stimulation ( D ) , specifically regulated upon BDNF stimulation ( E ) , bi - directionally regulated ( F ) . Expression of IR - transcripts spliced isoforms were analyzed by semi - quantitative PCR ( left panels ) . Means SDs of PIR values are shown beneath each panel . In addition , fold changes ( FC ) in expression of IR - transcripts ( red orange ) spliced isoforms ( grey ) were assessed with real - time qPCR .

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RT-PCR validations of IR-transcripts , specifically regulated upon bicuculline stimulation (D), specifically regulated upon BDNF stimulation (E), and bi-directionally regulated (F). Expression of the IR-transcripts and the spliced isoforms were analyzed by semi-quantitative PCR (left panels). Means and SDs of PIR values are shown beneath each panel. In addition, fold changes (FC) in the expression of the IR-transcripts (red and orange) and spliced isoforms (dark grey) were assessed with real-time qPCR.


A A representative photograph showing co - cultured DRG DH neurons . DRG neurons were expressed with Spy - pHluorin for imaging synaptic transmission . Scale bar , 10 μm . B of a presynaptic bouton marked in ( A ) showing Spy - pHluorin at 20 s before ( - 20 s , pre - stimulus ) , 20 s , 40 s , 120 s after electrical stimulation ( post - stimulus ) in 0Ca2 + ( upper panel ) or 2Ca2 + ( lower panel ) solution . C Averaged changes ( ΔF / F0 ) of Spy - pHluorin in 0Ca2 + ( left ) or 2Ca2 + solution ( right ) in response to same electrical stimulation ( 20 Hz , 20 s ) ( n = 45 from 6 for 0Ca2 + 57 from 6 for 2Ca2 + ) . shadow in traces represents error bars ( s . e . m ) of each point .

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A A representative photograph showing the co-cultured DRG and DH neurons. The DRG neurons were expressed with Spy-pHluorin for imaging the synaptic transmission. Scale bar, 10 μm. B Images of a presynaptic bouton marked in (A) showing the Spy-pHluorin fluorescence at 20 s before (-20 s, pre-stimulus), 20 s, 40 s, and 120 s after electrical stimulation (post-stimulus) in 0Ca2+ (upper panel) or 2Ca2+ (lower panel) solution. C Averaged fluorescence changes (ΔF/F0) of Spy-pHluorin in 0Ca2+ (left) or 2Ca2+ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from 6 cells for 0Ca2+ and 57 puncta from 6 cells for 2Ca2+). The shadow in the traces represents the error bars (s.e.m) of each point.


D - F same as in ( A - C ) , but experiments were performed in cultured hippocampal neurons ( n = 70 from 3 for 0Ca2 + 72 from 3 for 2Ca2 + ) .

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D-F The same as in (A-C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from 3 cells for 0Ca2+ and 72 puncta from 3 cells for 2Ca2+).


B Representative western blots ( upper ) ( lower ) of expression of Syb2 in control ( Ctrl ) Syb2 - cKO DRG neurons ( n = 3 per group ) . Control DRG neurons is from homozygous floxed Syb2 - null mice infected with AAV5 - CAG - EGFP .

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B Representative western blots (upper) and analysis (lower) of the expression levels of Syb2 in control (Ctrl) and Syb2-cKO DRG neurons (n = 3 per group). Control DRG neurons is from homozygous floxed Syb2-null mice infected with AAV5-CAG-EGFP virus.


C Evoked EPSCs statistics from DH neurons co - cultured with control ( Ctrl ) or Syb2 - cKO DRG ( cKO ) in 0Ca2 + solution ( n = 13 for Ctrl 11 for cKO ) .

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C Evoked EPSCs and statistics from DH neurons co-cultured with control (Ctrl) or Syb2-cKO DRG cells (cKO) in 0Ca2+ solution (n = 13 cells for Ctrl and 11 for cKO).


D Evoked EPSCs statistics from DH neurons co - cultured with DRG neurons before ( black ) after ( red ) applying 1 μM ω - conotoxin - GVIA ( GVIA ) in 0Ca2 + bath ( n = 9 ) .

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D Evoked EPSCs and statistics from DH neurons co-cultured with DRG neurons before (black) and after (red) applying 1 μM ω-conotoxin-GVIA (GVIA) in 0Ca2+ bath (n = 9 cells).


F Left panels , evoked EPSCs from DH neurons co - cultured with scrambled shRNA control ( Ctrl ) or Cav2 . 2 - KD ( sh - 1 / sh - 2 ) DRG neurons in 0Ca2 + solution . Right panel , of evoked EPSCs ( n = 17 for Ctrl , 12 for sh - 1 , 19 for sh - 2 ) . EPSCs were evoked by local electrical stimulation ( Estim , at arrows ) .

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F Left panels, evoked EPSCs from DH neurons co-cultured with scrambled shRNA control (Ctrl) or Cav2.2-KD (sh-1/sh-2) DRG neurons in 0Ca2+ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh-1, and 19 for sh-2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).


C Western blots of lysates of HEK293T , resolved on SDS - PAGE stained with a pan - arrestin antibody . mt = mock transfected . Bpa : Benzoyl - Phenylalanine . Arrestins were equipped with a 3xHA tag at C - terminus , which increases their of about 3 kDa compared to endogenous ( βarr1 47 kDa , βarr2 46 kDa ) . βarr1 βarr2 were transfected at 1 / 3 compared to corresponding TAG - mutants .

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C Western blots of the lysates of HEK293T cells, resolved on SDS-PAGE and stained with a pan-arrestin antibody. mt = mock transfected. Bpa: Benzoyl-Phenylalanine. Arrestins were equipped with a 3xHA tag at the C-terminus, which increases their size of about 3 kDa compared to the endogenous proteins (βarr1 47 kDa, βarr2 46 kDa). βarr1 and βarr2 were transfected at 1/3 DNA compared to the corresponding TAG-mutants.


Each panel represents immunoblots of whole lysates stained with an anti - HA antibody detecting arrestin . In left panels GPCRs were combined with βarr1 variants , in right panels with βarr2 variants . Residues replaced by Bpa are indicated above each lane . Covalent arrestin - receptor complexes are expected between 100 - 200 kDa , considering of receptors ( V2R 41 . 5 kDa , PTH1R 63 . 8 kDa , CRF1R , 46 . 3 kDa ) their glycosylation . A PTH1R , agonist PTH ( 1 - 34 ) B CRF1R , agonist Urocortin 1 ( Ucn1 ) C V2R , agonist arginine - vasopressin ( AVP ) D No receptor .

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Each panel represents immunoblots of whole cell lysates stained with an anti-HA antibody detecting arrestin. In the left panels GPCRs were combined with βarr1 variants, in the right panels with βarr2 variants. Residues replaced by Bpa are indicated above each lane. Covalent arrestin-receptor complexes are expected between 100-200 kDa, considering the size of the receptors (V2R 41.5 kDa, PTH1R 63.8 kDa, CRF1R, 46.3 kDa) and their glycosylation. A PTH1R, agonist PTH(1-34) B CRF1R, agonist Urocortin 1 (Ucn1) C V2R, agonist arginine-vasopressin (AVP) D No receptor.


Aliquots from agonist - treated samples shown in Figure 2 were deglycosylated with PNGaseF , run on SDS - PAGE immunoblotted with antibodies detecting arrestin ( upper panels , α - HA ) GPCRs ( as indicated , CRF1R is equipped with a N - terminal FLAG , V2R carries a C - terminal 1D4 epitope ) .

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Aliquots from agonist-treated samples shown in Figure 2 were deglycosylated with PNGaseF, run on SDS-PAGE and immunoblotted with antibodies detecting arrestin (upper panels, α-HA) and the GPCRs (as indicated, CRF1R is equipped with a N-terminal FLAG, V2R carries a C-terminal 1D4 epitope).


A SDS - PAGE immunoblots of immunoprecipitated samples . Sample 1 was immunoprecipitated without previous UV treatment . Sample 2 is product of irradiation of sample 1 . Sample 3 was treated with UV before immunoprecipitation . samples were run in parallel on same gel .

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A SDS-PAGE and immunoblots of immunoprecipitated samples. Sample 1 was immunoprecipitated without previous UV treatment. Sample 2 is the product of irradiation of sample 1. Sample 3 was treated with UV light before immunoprecipitation. All samples were run in parallel on the same gel.


B exclusion chromatography ( SEC ) of Sample 3 .

B Size exclusion chromatography (SEC) of Sample 3.


C Western blot of fractions eluted by SEC .

C Western blot of the fractions eluted by SEC.


MALDI - TOF of SEC fractions VR2 VR3 . spectrum of sample VR2 shows a [ M + H ] + peak featuring twice mass ( ~ 108 000 Da ) of [ M + H ] + peak of βarr1 monomer ( sample VR3 , ~ 50 400 Da ) .

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MALDI-TOF analysis of the SEC fractions VR2 and VR3. The spectrum of sample VR2 shows a [M+H]+ peak featuring twice the mass (~108 000 Da) of the [M+H]+ peak of the βarr1 monomer (sample VR3, ~50 400 Da).


C Western blot of whole lysates from HEK293T expressing βarr1 mutated as indicated in top lines . samples were run in parallel on same gel .

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C Western blot of whole cell lysates from HEK293T cells expressing βarr1 mutated as indicated in the top lines. All samples were run in parallel on the same gel.


( E ) Western blot of whole lysates stained with an α - HA antibody . Residues exchanged with BrEtY are indicated in upper row , mutations at PTH1R are indicated on left side . red arrows indicate two most prominent signals . Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R .

(E) Western blot of whole cells lysates stained with an α-HA antibody. Residues exchanged with BrEtY are indicated in the upper row, mutations at PTH1R are indicated on the left side. The red arrows indicate the two most prominent signals. Red squares indicate signals that vanish upon removal of specific Cys residues in PTH1R.


A . Western blot detection of PEX13 , ATG7 , SMURF1 , FANCC in HeLa / GFP - LC3cells transfected with indicated siRNA . Asterisk denotes non - specific band .

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A. Western blot detection of PEX13, ATG7, SMURF1, and FANCC in HeLa/GFP-LC3cells transfected with the indicated siRNA. Asterisk denotes non-specific band.


B . Representative of GFP - LC3 colocalization with mCherry - capsid at 10 h after Sindbis ( AO30 ) infection of HeLa / GFP - LC3 treated with indicated siRNA . siPEX13 oligo # 2 is shown ; similar results were observed with three other siPEX13oligos . Arrowheads denote representative colocalized GFP - LC3 / mCherry - capsid . Scale bars , 10 μm .

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B. Representative images of GFP-LC3 colocalization with mCherry-capsid at 10 h after Sindbis virus (strain AO30) infection of HeLa/GFP-LC3 cells treated with the indicated siRNA. siPEX13 oligo #2 is shown; similar results were observed with three other siPEX13oligos. Arrowheads denote representative colocalized GFP-LC3/mCherry-capsid puncta. Scale bars, 10 μm.


C . of colocalized GFP - LC3 mCherry - capsid normalized to number of mCherry - capsid per ( % colocalization ) in experiment shown in ( B ) . Bars are mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in more than three independent experiments . * * * p < 0 . 001 ; one - way ANOVA with adjustment for multiple comparisons . See Fig EV1A B for of mCherry - capsid per GFP - LC3 per from same experiment .

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C. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) in the experiment shown in (B). Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. ***p<0.001; one-way ANOVA with adjustment for multiple comparisons. See Fig EV1A and B for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.


D . Western blot detection of Pex13 in MEFs of indicated genotype . Asterisks denote non - specific bands .

D. Western blot detection of Pex13 in MEFs of indicated genotype. Asterisks denote non-specific bands.


E . of colocalized GFP - LC3 mCherry - capsid normalized to number of mCherry - capsid per ( % colocalization ) at 8 h after Sindbis ( AO28 ) infection in MEFs of indicated genotype . Bars are mean ± of triplicate samples ( ~ 100 analyzed per sample ) . Similar results were observed in more than three independent experiments . * p < 0 . 05 ; two - unpaired t - . See Fig EV1G H for of mCherry - capsid per GFP - LC3 per from same experiment .

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E. Quantification of colocalized GFP-LC3 and mCherry-capsid puncta normalized to the number of mCherry-capsid puncta per cell (% colocalization) at 8 h after Sindbis virus (AO28 strain) infection in MEFs of the indicated genotype. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). Similar results were observed in more than three independent experiments. *p<0.05; two-tailed unpaired t-test. See Fig EV1G and H for quantification of mCherry-capsid puncta per cell and GFP-LC3 puncta per cell from the same experiment.


F . Western blot detection of p62 LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle cultured in normal medium ( starvation " - " ) or EBSS ( starvation " + " ) for 3 h . Similar results were observed in three independent experiments .

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F. Western blot detection of p62 and LC3 in MEFs of indicated genotype treated with 100 nM Baf A1 or DMSO vehicle and cultured in normal medium (starvation "-") or EBSS (starvation "+") for 3 h. Similar results were observed in three independent experiments.


G . of GFP - LC3 in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle cultured in normal medium or HBSS ( starvation " + " ) for 3 h . Bars are mean ± of triplicate samples ( ~ 100 analyzed per sample ) . * p < 0 . 05 ; two - unpaired t - . NC , nonsilencing control .

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G. Quantification of GFP-LC3 puncta in MEFs of indicated genotype treated with 10 nM Baf A1 or DMSO vehicle and cultured in normal medium or HBSS (starvation "+") for 3 h. Bars are mean ± SEM of triplicate samples (~100 cells analyzed per sample). *p<0.05; two-tailed unpaired t-test. NC, nonsilencing control.


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 .

(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 .

(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 .

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 .

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 .

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.