EMBO/soda-nel huggingface dataset:
Retinoic acid-induced type 1 regulatory T cells suppress autoimmunity (2019)
Figure 1

Figure 1. Immunization with antigen, RA and IL-2 induces antigen-specific IL-10-secreting LAG-3+ CD4 T cells.

A Ex vivo flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + IL-2, KLH + RA, KLH + RA + IL-2. Results are representative FACS plots of LAG3+CD4+ or CD25+Foxp3+ cells after gating on live single CD3+CD4+CD49b+Foxp3- and live single CD3+CD4+ lymphocytes respectively.

B Mean absolute numbers of LAG-3+CD49b+Foxp3-or CD25+Foxp3+ CD4 T cells in LNs as described in (A). Bars are mean + SEM (n=3, 4 or 5) *p<0.05 by one-way ANOVA with Tukey post hoc test.

C Expression of Tr1 markers PD-1, CD226, ICOS, CD25 and CTLA-4 on CD49b+ LAG-3+ Foxp3-CD4+ T cells or on CD49b-CD4+ T cells.

D IL-10, IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice immunized with PBS + DMSO (Veh), KLH + DMSO (KLH), KLH + RA, KLH + IL-2, KLH + RA + IL-2 or KLH + LP-GMP (LP1569 + c-di-GMP) and cultured for 5 days with increasing concentrations of KLH. Bars are mean + SEM (n=4 or 5). **p<0.01, ***p<0.001, KLH + IL-2 + RA vs KLH, ##p<0.01, ###p <0.001 KLH + IL-2 + RA vs KLH + RA, †††p<0.001 KLH + IL-2 + RA vs KLH + IL-2 by two-way ANOVA and Tukey post hoc test.
Retinoic acid-induced type 1 regulatory T cells suppress autoimmunity (2019)
Figure 2

Figure 2. Induction of Tr1 cells by immunization of mice with MOG, RA and IL-2 protects against the development of EAE.

A Flow cytometry on LN cells of mice 7 days after two s.c. immunizations (14 days apart) with PBS + DMSO (Veh), MOG + DMSO (MOG), MOG + RA + IL-2. Cells were gated on live single CD3+ lymphocytes. FMO: Fluorescence Minus One.

B Mean percentages of IL-10+, LAG-3+, PD1+ or Foxp3+ CD25+ CD4 T cells as described in A. Cells were gated on live single CD3+ lymphocytes (top left panel) or on live single CD3+ CD4+ lymphocytes. Bars are mean + SEM (n=5). *p <0.05, ***p <0.01 by one-way ANOVA and Tukey post hoc test.

C EAE clinical scores in mice immunized s.c. with MOG + RA, MOG + IL-2, MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=12, except for MOG + IL-2, where n=6; combined from two experiments). *p<0.05, **p<0.01, ***p <0.001, MOG + IL-2 + RA vs Veh, #p<0.05, ##p<0.01, ###p<0.001, MOG + IL-2 + RA vs MOG + RA, †p<0.05, ††p<0.01, †††p <0.001, MOG + IL-2 + RA vs MOG + IL-2 by two-way ANOVA and Tukey post hoc test.

D Representative FACS plots of MOG dextramer+ CD4 T cells in LNs of naïve mice or 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+.

E Mean percentages of MOG dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+. Bars are mean + SEM (n=5 or 6). *p <0.05 by unpaired two-tailed t test.

F Mean fluorescence intensity (MFI) of CD49b, ICOS and PD-1 by MOG-dextramer+ CD4 T cells in LNs 3 days after induction of EAE. Cells were gated as single live F4/80- CD8- B220- CD4+ CD44+ dextramer+. Bars are mean + SEM (n=5 or 6). *p <0.05, **p <0.01 by unpaired two-tailed t test.

G MOG-specific IL-17 and IFN-γ production by spleen cells from day 7 of EAE, quantified in supernatants by ELISA after 5 d culture with increasing concentrations of MOG. Bars are mean + SEM (n=3 or 4). *p<0.05, **p<0.01 and ***p<0.001 by two-ANOVA and Tukey post hoc test.
Retinoic acid-induced type 1 regulatory T cells suppress autoimmunity (2019)
Figure 3

Figure 3. Suppressed pathogenic Th17 responses correlate with LAG-3+ CD4 T cells.

A Representative FACS plots of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2 or vehicle (Veh) only. Cells were gated as single live CD45+ CD3+ CD4+.

B Mean absolute numbers of CD11a+ CD4 T cells in the spleen on day 7 of EAE in mice immunized with MOG, RA and IL-2. Bars are mean + SEM (n=4 or 5). *p<0.05 by unpaired two-tailed t test.

C Representative FACS plots of CD45+ CD4 T cells in the spinal cords of mice on day 12 of EAE. Cells were gated as single live CD45+.

D Mean absolute numbers of infiltrating CD45+ cells in the spinal cords of mice on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.

E Representative FACS plots of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.

F Mean absolute numbers of infiltrating IL-17+ and IFN-γ+ CD4 T cells in the spinal cords on day 12 of EAE. Bars are mean + SEM (n=5 or 6). *p<0.05 by unpaired two-tailed t test.

G Representative histogram of LAG-3 expression on CD4 T cells in the spinal cords on day 12 of EAE. Cells were gated as single live CD45+ CD3+ CD4+.

H Inverse correlation between LAG-3 and IL-17 and IFN-γ expression on CD4 T cells in the spinal cords on day 12 of EAE. *p<0.05 by unpaired two-tailed t test.
Retinoic acid-induced type 1 regulatory T cells suppress autoimmunity (2019)
Figure 4

Figure 4. MOG-specific Tr1 cells suppress pathogenic Th1 and Th17 cells.

A IL-17 and IFN-γ production by ELISA on supernatants of spleen cells from mice with EAE (EAE) co-cultured for 3 days with MOG-specific Tr1 cells (from mice immunized twice with MOG, RA and IL-2 and amplified in vitro with MOG, RA and IL-2 for 8 days) or control T cells from LNs of mice injected with PBS + DMSO (Veh). Bars are mean + SD of one experiment. *p<0.05, **p<0.01 and ***p<0.001 by unpaired two-tailed t test.

B EAE clinical scores of mice transferred 1 day before induction of EAE with MOG-specific Tr1 cells amplified in vitro with MOG, RA and IL-2. Results are mean + SEM (n=4 or 6). *p<0.05 and **p<0.01 by two-way ANOVA with Bonferroni post hoc test.

C Representative FACS plots of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Cells were gated as live single CD45.2+CD3+CD4+ cells.

D Mean absolute numbers of IL-17+ CD4 T cells in the spinal cords on day 20 of EAE. Results are mean + SEM (n=6). *p<0.05 by unpaired two-tailed t test.

E Representative FACS plots of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Cells were gated as live single CD45.1+CD3+CD4+.

F Mean percentages of CTLA-4+ CD4 T cells in the spleens of mice with EAE. Results are mean + SEM (n=6). **p<0.01 by unpaired two-tailed t test.

G EAE clinical scores of WT and IL-10-/- mice immunized s.c. with MOG + RA + IL-2, or PBS + DMSO (Veh), 7 and 21 days before induction of EAE. Results are mean + SEM (n=6 or 7). ***p <0.001, WT MOG + IL-2 + RA vs WT Veh, ##p<0.01, ###p<0.001, IL-10-/- MOG + IL-2 + RA vs IL-10-/- Veh by two-way ANOVA and Tukey post hoc test.
Retinoic acid-induced type 1 regulatory T cells suppress autoimmunity (2019)
Figure 5

Figure 5. Induction of Tr1 cells by immunization of mice with autoantigen, RA and IL-2 protects against EAU and spontaneous uveitis.

A Retinal inflammatory scores of mice immunized s.c. with PBS + DMSO (Veh), IRBP + DMSO (IRBP) or IRBP + RA + IL-2 21 and 7 days before induction of EAU by s.c. injection of IRBP emulsified in CFA. Bars are mean + SEM (n=6), *p<0.05 and ***p<0.001, IRBP + IL-2 + RA vs Veh, ###p<0.001 IRBP + IL-2 + RA vs IRBP by two-way ANOVA with Tukey post hoc test.

B Representative fundus images of each group of mice 35 days after induction of EAU. Arrowheads indicate area with retinal vasculitis and the asterisk indicates a region of vitreous haze, where the image appears blurred.

C Retinal inflammation scores in HEL-TCR transgenic mice were s.c. injected with PBS + DMSO (Veh) or HEL + RA + IL-2 on pn 18 (1x) or on pn 18 and 25 (2x). Results are mean + SEM (n=3, 4 or 5). *p<0.05 and ***p<0.001, HEL + IL-2 + RA 1x vs Veh, ##p<0.01 HEL + IL-2 + RA 2x vs Veh by two-way ANOVA with Tukey post hoc test.

D Representative photographs of each group of retinal fundus of HEL-TCR transgenic mice at pn 35. Arrowheads indicate representative areas with retinal vasculitis.

E Representative FACS plots of IL-17+ CD4 or IL-10+ CD4 T cells in the retina on day 36 of HEL-TCR transgenic mice. Cells were gated on live single CD3+CD4+ and CD3+CD4+LAG-3+Foxp3- respectively.

F Mean absolute number of IL-17+ CD4 T cells in the retina (upper panels) or mean percentages of IL-10+ LAG-3+ Foxp3- CD4 T cells in the spleens of of HEL-TCR transgenic mice with vasculitis (lower panels). Results are mean + SEM (n=3, 4 or 5). **p <0.01 and ***p <0.001 by one way ANOVA with Bonferroni post hoc test.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 1

Figure 1. MLKL undergoes ubiquitylation during necroptosis.

A WT MDFs were treated ± TSI individually or in combination for 3 hrs. Whole cell lysates (WCL) and UBA-pull down (UBA-PD) fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments. Samples of UBA-pull down in following figures were analysed in the same way unless otherwise indicated.

B WT, Ripk3-/-, Mlkl-/-, Tnfr1-/- and Traf2-/- MDFs were untreated (UT) or treated with TSI for 3 hrs. Nec-1 and GSK872 were added to inhibit RIPK1 and RIPK3 kinase activities respectively.

C WT and Tnf-/- BMDMs were treated ± death ligands including TNF (T), LPS (L), Fas ligand (F) and Poly I:C (P) in addition to S and I for 3 hrs.

D WT HT29 cells were untreated (UT) or treated with TSI for 16 hrs.

E RIPK3-gyrase were inducibly expressed in Ripk3-/- MDFs by doxycycline (dox) for 5 hrs, and cells were then treated ± combination of TSI, or coumermycin (coum) for 3 hrs.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 3

Figure 3. MLKL ubiquitylation accumulates in the crude membrane fraction and can be digested by USP21 located on biological membranes.

A WT MDFs were treated with TSI for indicated time. Cells were fractionated into cytosol and crude membrane, followed by UBA-pull down. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.

B WT USP21-CaaX and USP21C221R-CaaX (CR) were inducibly expressed in WT MDFs by doxycycline for 5 hrs. Ubiquitylated proteins were enriched followed by TSI stimulation and cellular fractionation. All fractions were analysed by Western blot and probed with antibodies as indicated. Representative of three independent experiments.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 4

Figure 4. MLKL oligomerization drives its necroptosis specific ubiquitylation.

A WT and R105AD106A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were untreated (UT) or treated with TSQ for 6 hrs. Cells were fractionated into cytosol (C) and crude membrane (M). Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments.

B Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.

C WT, Q343A and S345D mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, at the same time cells were treated ± TSQ for 16 hrs, followed by UBA-pulldown. Representative of three independent experiments.

D WT and Q343A mutant MLKL were inducibly expressed in Ripk3-/-Mlkl-/- MDFs by doxycycline, at the same time cells were treated ± TSQ for 16 hrs, followed by UBA-pulldown. Representative of three independent experiments.

E HT29 cells were stimulated with TSI, ± NSA (500 nM), or left untreated (UT) for 16 hrs, followed by cellular fractionation. Fractions were analysed by BN- or SDS-PAGE, Western blot and probed with the indicated antibodies. Representative of three independent experiments.

F Cell lysates from (E) were subjected to UBA-pulldown and analysed as described above.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 5

Figure 5. MLKL-ubiquitylation does not lead to cell death, but correlates with the turnover of activated MLKL.

A WT and E109AE110A mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline, cells were untreated or treated with TSQ for 6 hrs (please note that the same WT control was used in Fig. EV3A). Cell death was measured by PI staining based on flow cytometry. Data are plotted as mean ± SEM of three independent experiments.

B Cellular fractions from (A) were analysed by Western blot from BN-PAGE or SDS-PAGE using antibodies as indicated. The same WT control was used in Fig. 4A. Representative of three independent experiments.

C Cell lysates from (A) were subjected to UBA-pull down and analysed as described above.

D N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline overnight, and cells were treated with TSI for indicated time, followed by UBA-pull down. Representative of three independent experiments.

E N-FLAG MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 16 hrs. Cells were stimulated ± TSI for 3 hrs after withdrawal of doxycycline. Then TSI medium was removed and replaced to medium containing inhibitors Bafilomycin A1 (BAF), PS341 (PS) or left untreated (UT). IDN-6556 was added to all conditions to block apoptosis. Cells were collected 0, 2, 4, 6 hrs after medium replacement, followed by UBA-pull down. Representative of three independent experiments.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 6

Figure 6. Simultaneous arginine replacement of 4 ubiquitylation sites on the mMLKL 4HB domain does not prevent necroptosis-induced ubiquitylation.

A Cartoon of the N-terminal region (residues 1-180) of mouse MLKL (PDB accession 4BTF;(Murphy, Czabotar et al. 2013)) showing the four lysine residues identified from MS analysis as yellow sticks.

B WT and 4KR mutant MLKL were inducibly expressed in Mlkl-/- MDFs by doxycycline for 6 hrs and cells were untreated (UT) or treated with TSI, followed by UBA-pull down. Representative of three independent experiments.
Oligomerisation-driven MLKL ubiquitylation antagonises necroptosis (2021)
Figure 7

Figure 7. MLKL ubiquitylation antagonises necroptosis.

A Schematic of the MLKL-USP21 and USP21 catalytic dead mutant proteins.

B Mouse MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in Mlkl-/- MDFs by doxycycline (10 ng/mL) for 6 hrs with addition of a necroptotic stimulus (TSI) for the indicated time, followed by UBA-pulldown. Non-specific band is indicated with "*" in the blot of RIPK3. Representative of three independent experiments.

C Mlkl-/- MDFs and Ripk3-/-Mlkl-/- MDFs stably transfected with constructs encoding MLKL, USP21, USP21C221R, MLKL-USP21 and MLKL-USP21C221R were treated with doxycycline, TSI or in combination. Propidium iodide positive cells were quantified in real time by IncuCyte live cell imaging. Data are plotted as mean ± SEM of three independent experiments.

D MLKL-/- HT29 cells stably transfected with constructs encoding human MLKL-USP21 and MLKL-USP21C221R, were treated with doxycycline, NSA (1 μM), TSI or combinations thereof (added simultaneously). Sytox Green positive cells were quantified in real time by live cell imaging. Data are plotted as mean ± SEM of six independent experiments. (A red dashed line is shown to highlight the delay in death kinetics upon treatment with NSA).

E Human MLKL-USP21 and MLKL-USP21C221R were inducibly expressed in MLKL-/- HT29 cells by doxycycline (10 ng/mL) for 16 hrs, TSI was added for the indicated times but all samples were collected 25 hrs post-induction, followed by UBA-pulldown. Representative of three independent experiments.

Expanded view figure legends
Changing POU dimerization preferences converts Oct6 into a pluripotency inducer (2016)
Figure 1

Figure 1. Oct transcription factors exhibit differential preferences for composite DNA binding sites.

A. Position weight matrices obtained using de novo motif discovery (HOMER) and ChIP-Seq summits of Oct4 in mouse ESCs [56], Brn2 in MEFs 48 hours after transfections and Brn2 in mouse NPCs [23].

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 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.
Changing POU dimerization preferences converts Oct6 into a pluripotency inducer (2016)
Figure 2

Figure 2. A single amino acid residue guides DNA-binding preferences of Oct4 and Oct6 POU domains.

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. 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.

G. A scheme illustrating the DNA binding preferences of Oct POU domains to MORE and SoxOct elements. On the left side, WT Oct4 (green) preferentially heterodimerize with Sox2 HMG (cyan) over homodimerizing on MORE. In the second scenario, WT Oct6 (orange) preferentially forms homodimers on MORE DNA rather than heterodimers with Sox2 on SoxOct DNA elements. The thickness of the arrows illustrates the relative abundance of the microstates under equilibrium conditions. The binding behavior is swapped for the engineered Oct6151S and Oct4151M proteins.
Changing POU dimerization preferences converts Oct6 into a pluripotency inducer (2016)
Figure 3

Figure 3. The residue guiding DNA-binding preference is a key Oct4 determinant in pluripotent cell generation.

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.

C. On the left side, a schematic overview of Oct4 and its mutants used for iPSC generation. POUS and POUHD are shown as green bars connected by the linker. Sites mutated to the respective Oct6 residues and Oct6 linker are denoted in orange, and the mutation in the Sox-Oct interface is in black. The efficiency of these constructs for iPSC generation from MEFs is depicted as the absolute number of GFP-positive colonies on the right. Error bars represents standard deviations of biological replicates (n=3) and differences between selected samples were compared using ANOVA (p-values: Oct4WTxOct47D,22K p = 6.9e-3; Oct4WTxOct4LinkO6 p = 7.3e-4; Oct4WTxOct4151M p = 1.2e-2; Oct4151MxOct4LinkO6,151M p = 1.1e-2; Oct4LinkO6,151M xOct47D,22K,LinkO6,151M p = 7.1e-3) (*** p < 0.001, ** p < 0.01, * p < 0.05).

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.
Changing POU dimerization preferences converts Oct6 into a pluripotency inducer (2016)
Figure 4

Figure 4. The synthetic Oct6 molecule contributes to epigenetic reprogramming of mouse embryonic fibroblasts.

A. On the left side, a schematic overview of Oct6 and its mutants used for iPSC generation. POUS and POUHD are shown as orange bars connected by the linker. Sites mutated to the respective Oct4 residues and Oct4 linker are denoted in green, and the mutation in the Sox-Oct interface is in black. The efficiency of these constructs for iPSC generation from MEFs is depicted as the absolute number of GFP-positive colonies on the right. Error bars represents standard deviations of two biological replicates run in parallel.

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 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.