Fig 2: FACT Depletion Increases Efficiency of Reprogramming Human Fibroblasts(A) Reprogramming with human secondary fibroblasts carrying a doxycycline (DOX)-inducible OCT4/SOX2/KLF4/c-MYC (OSKM) cassette (Cacchiarelli et al., 2015) that were transfected with siRNAs against human SSRP1 or SUPTH16 before DOX induction.(B) Alkaline phosphatase (AP) staining of stem cell colonies 21 days after DOX treatment. Control experiment is scrambled siRNA (see STAR Methods).(C) Quantification of iPSC colonies (6 replicates) based on SSEA4 immunostaining for each knockdown condition. Paired Student’s t test was used for statistical comparison, ∗p < 0.05, ∗∗∗ p < 0.001. Error bars represent SD.(D) Histological characterization of teratomas derived from grafting SSRP1 or SUPT16H depletion-derived iPSCs in mice. Teratomas reached 1.5 cm2 in size after 51–70 days and showed tissues of all three germ layers (ectoderm, endoderm, mesoderm). Scale bars represent 50 μm.(E) Forced expression of Ascl1, Brn2, and Mytl1 in human fibroblasts (Normal Human Dermal Fibroblats = NHDF cells) and SSRP1 or SUPT16H depletion enhances reprogramming of fibroblasts to neurons. One-way ANOVA test was used for statistical comparison, ∗∗ p < 0.01. Error bars represent SEM.See also Figure S5.

Fig 3: CPF suppresses transcription of FACT and c‐MYC genes. A, Quiescent A549 cells were transfected with either empty vector (control), or FACT plasmid (SPT16+ or SSRP1+), or treated with CPF at GI90, or a combination of FACT plasmid and CPF at GI90 (rescue) upon cell cycle re‐entry for 36h. The cells were harvested for cell cycle analysis. *P < .05 vs control; B, ChIP was performed in the presence of CPF at GI90 or DMSO for 36 h following release from 3‐day contact inhibition in A549 cells. PCR primers were designed based on FACT and c‐MYC promoter or control region. *P < .05 vs control. C, Knockdown of c‐MYC with three sets of siRNA and its impact on FACT mRNA and protein levels by RT‐PCR, immunoblotting and quantification. *P < .05 vs NC control

Fig 4: SSRP1-Q265K mutation reduces FACT’s turnover rate on chromatin(A) Representative images of SPT16-GFP signals in wild-type or Ssrp1Q265K cells. The white dashed circle indicates the bleached area. Scale bar, 5μm.(B) Quantitative analysis of relative Spt16-GFP fluorescence intensity of the bleached areas over time. The data are presented as mean ± SEM.(C) Time for 50% fluorescence intensity recovery after photobleaching (T1/2). Center lines show the median; box limits indicate the 1st and 3rd quartiles; the bars represent the minimum and maximum values. Each dark spot represents the individual result. The numbers above the boxplots represent the mean T1/2 with SD. n represents the number of data values (unpaired two-tailed Student’s t test was performed, ∗∗∗∗p < 0.0001).

Fig 5: CPF treatment decreases FACT mRNA and protein levels. Upon release from quiescence, A549 and H1975 cells were treated with CPF at GI90 for 6‐24 h and then harvested for RT‐qPCR (A) or immunoblotting (B) for SPT16 and SSRP1. P: proliferative cells, Q: quiescent cells; R: release from quiescence; C: control; and T: CPF treatment. *P < .05 vs control at same time‐point. C, The quiescent cells were exposed to CPF at GI90 for the indicated time from releasing from quiescence and analysed for p27 and its degradation proteins by immunoblotting. *P < .05 vs control at same time‐point