Fig 2: Phase and subpopulation specific p38MAPK signalling during reprogramming of human fibroblasts.(a) Quantitave RT-PCR analysis of MKK3, MKK6 and p38 MAPK expression in H9 (p35), neonatal human fibroblasts (Neo1), adult human fibroblasts (Ad3) and iPSC generated therefrom (Neo1 clone 2, Neo1 clone 5, Neo1 clone 6, Neo1 clone 7, Ad3 clone 2, Ad3 clone 2 and Ad3 clone 7). Data represent relative expression to GAPDH and were normalized against H9. Data are presented as mean ± S.E.M. Student t-test was carried out to detect significant changes between hESCs (H9) and neonatal and adult fibroblasts and respective iPSC. *p < 0.05; **p < 0.01; (b) Western blot analysis showing expression of the total form of p38MAPK and phospho p38 (Thr180/Tyr182) MAPK in hESC (H9), neonatal human fibroblasts (Neo1), adult human fibroblasts (Ad3) at Day 0 and iPSC derived therefrom. Cropped images of at least three repeats are shown for purpose of clarity; (c) Western blot analysis showing expression of the total form of p38MAPK and phospho-p38 (Thr180/Tyr182) MAPK during the time course of reprogramming of neonatal (Neo1) and adult (Ad3) fibroblasts. Days of transduction = D. GAPDH served as loading control. Images are representative of at least three independent experiments and are cropped; (d) Graphic representation of the percentage of p-p38MAPK positive cells at different cellular populations (TRA1-60 + /CD44-, TRA1-60 + /CD44 + and TRA1-60-/CD44 + ) during the reprogramming of neonatal (Neo1) fibroblasts assessed by flow cytometric analysis. Data are presented as mean ± S.E.M, n = 3.

Fig 3: Application of p38MAPK inhibitor (SB202190) abrogates iPSC generation.(a) Western blot analysis of p-p38MAPK downregulation by SB202190 (p38MAPKi) in hESCs (H9). GAPDH is used as a loading control. Images are representative of at least three independent experiments. Cropped images of at least three repeats are shown for purpose of clarity; (b) Schematic representation of inhibitor application (p38i) at Day 8 during the reprogramming process; (c) Phase–contrast observation showing the morphology of partially reprogrammed colonies arising during the reprogramming of Neo1 fibroblasts treated with DMSO or p38MAPKi for 24 hours at Day 8, scale bars = 100 µm; (d) Graphic representation of flow cytometric data analysis indicating a significant impact of p38MAPKi (i) application on the percentage of TRA1-60 + /CD44-, TRA1-60+/CD44 + and TRA1-60-/CD44 + cells at Days 10, 13 and 16. Results are presented as mean ± S.E.M, C: Control; i: p38MAPK inhibitor. Student t-test was carried out to detect significant changes between Control and p38MAPKi group, *p < 0.05; (e) Graphic representation of the colony numbers at Days 16 and 28 of reprogramming in p38MAPKi and DMSO treated Neo1 and Ad3 fibroblasts. Data are presented as mean ± S.E.M, n = 3. Student t-test was carried out to detect significant changes between Control and p38MAPKi group, *p < 0.05; (f) Alkaline phosphatase staining at Day 16 confirmed the absence of a large AP+ colonies with a typical morphology from Neo1 and Ad3 fibroblasts undergoing reprogramming and treated with p38MAPKi at Day 8 of transduction for 24 hours.

Fig 4: Inhibition of p38a MAPK signalling prevents generation of human iPSCs.(a) and (b) Assessment of p38a MAPK downregulation in neonatal (A) and adult (B) fibroblasts by quantitative RT-PCR analysis. Data represent relative expression to GAPDH, normalized against Control shRNA. Results are presented as average ± S.E.M, n = 3, Student t-test was carried out to detect significant changes between Control shRNA and p38a shRNA group, *p < 0.05; (c) Confirmation of p38a MAPK downregulation by quantitative RT-PCR analysis during reprogramming of Neo1 fibroblast at days 1 and 7. Data represent relative expression to GAPDH, normalized against Control shRNA. Results are presented as average ± S.E.M, n = 3, *p < 0.05; (c’) Western blot analysis of p38MAPK downregulation by RNAi in p38a shRNA transduced Neo1 fibroblasts at day 7. GAPDH is used as a loading control. Images are representative of at least three independent experiments. Cropped images of at least three repeats are shown for purpose of clarity; (d) Phase-contrast images of the Neo1 fibroblasts morphology treated with Control shRNA and p38a shRNA at day 0 and colonies arising during reprogramming of the control and p38a MAPK deficient Neo1 fibroblasts at Days 11 and 18. Scale bar = 100µm; (e) Graphic representation of flow cytometric data analysis indicating a significant impact of p38a shRNA downregulation on the percentage of TRA1-60 + /CD44- and TRA1-60 + /CD44 + cells at Days 13 and 18. Results are presented as mean ± S.E.M, n = 3. Student t-test was carried out to detect significant changes between Control shRNA and p38a shRNA group, *p < 0.05.