EMBO/SourceData huggingface dataset:
Figure 4(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) 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 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 images of mouse skin tumor sections labelled with antibodies to Plet1 and Atp6v1c2. Data information: D) Scale bars: 100 μm.
Figure 5(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 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 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 and semi-quantitative analysis Data information: Scale bar: 250 μm.
Figure 6(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.
Figure 7(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-test.(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 the mean expression level of MMR genes (measured in TPM) in Gata6wt (n=10) and Gata6mut (n=3) SebC.
figf1(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 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 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.
figf2(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).
figf3(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 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 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 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.
Figure 1B. 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 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. 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 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.
Figure 2A. 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. 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 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 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 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.
Figure 3A 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, and c-Myc. Colonies were imaged 16 days after second viral infection, using a fluorescence microscope. Scale bars: 250 μm; objective 10x.
Figure 4B. 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 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 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 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, 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 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 and O4SK iPSCs, illustrating the close relationship between their global gene expression profiles.
Figure 5A. 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 male mice with contribution from O6SKM iPSCs and CD1 female mice.C. Pictures demonstrating germline transmission; endogenous Oct4-GFP signal was detected in the gonads of F1 pups. Scale bars: 200 μm.
Figure 1(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. (
Figure 2(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 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) 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.
Figure 3(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) 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 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.
Figure 4(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 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).
Figure 5(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.
Figure 6(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) 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) 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.
Figure 7(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) 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.
Figure 1(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, and immunoblotted for LAMP1, EEA1, Rab11, Rab8, PMP70, GM130, and Na+/K+ ATPase.(D) Quantification of total cholesterol in the subcellular fractions Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.(E) Cells transfected with TMEM135 siRNA were subjected to a SREBP2 cleavage assay for the precursor of SREBP2 and nuclear SREBP2.(F) Cells transfected with TMEM135 siRNA were subjected to qPCR. Data represent mean ± SD (n=3 experiments), *P < 0.05, Student's t-test.
Figure 2(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) Cells were transfected with siRNAs as indicated and incubated with HPβCD in serum-starvation media followed by filipin staining. Scale bar, 20 μm.(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) 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.