Journal: bioRxiv

Article Title: FACT sets a barrier for cell fate reprogramming in C. elegans and Human

doi: 10.1101/185116

Figure Lengend Snippet: (A) Schematic showing procedure with human secondary fibroblasts carrying a doxycycline (DOX)-inducible, poly-cistronic human OCT4/SOX2/KLF4/c-MYC (OSKM) cassette that were transfected with siRNAs against human FACT subunit encoding genes SSRP1 and SUPTH16 before DOX induction. (B) Alkaline phosphatase (AP) staining of stem cell colonies 21 days after DOX treatment. Control experiment is scrambled siRNA (see Methods). (C) Quantification of iPSC colonies from 6 replications based on SSEA4 immunostaining as shown in for each knock-down condition. Paired student t test was used for statistical comparison, *p<0.05, **p<0.01, *** p<0.001. (D) Histological characterization of teratomas derived from grafting SSRP1 or SUPT16H depletion-derived iPSCs in mice. Teratomas reached 1.5 cm 2 in size on average after 51 days (SSRP RNAi iPSCs) or 70 days (SUPT16H RNAi ) and show tissues of all three germ layers (ectoderm, endoderm, mesoderm). Scale bars, 50 μm (E) Forced expression of the TFs Ascl1, Brn2, Mytl1 (BAM) in human fibroblasts (NHDF cells) and SSRP1 or SUPT16H depletion enhances reprogramming of fibroblasts to neurons by approximately 50%. Beta-III tubulin-positive neurons derived from FACT-depleted fibroblasts showed a higher degree of projection complexity compared to control cells. One-way ANOVA test was used for statistical comparison, ** p<0.01. (F) ATAC-seq was performed in hiF-T cells without OSKM induction 48h after FACT depletion. Hexbin density scatterplot of SSRP1 RNAi normalized log2 fold-changes plotted against SUPT16H RNAi normalized log2 fold-changes for those ATAC-seq peaks that were significantly differential (q < 0.01) in at least one of the two conditions. (G) De novo motif generation (see Material and Methods) in opening and closing regions upon SSRP1 RNAi and SUPT 16H RNAi . Top 3 enriched motifs for each indicated set are given.

Article Snippet: Induction of OSKM by DOX supplementation (2μg/ml) was started the next day in hiF medium for the first 2 days and then in KSR medium (DMEM/F12 Gltuamax supplemented with 20% KSR, 1% NEAA, 0, 1% beta-mercaptoethanol 100 U ml -1 penicillin, 100 μg ml -1 streptomycin, 8 ng/ml FGFbasic and ROCK1 inhibitor (Y-27632-2HCl, Biozol Diagnostica, final concentration 1μM)) as described previously ( ).

Techniques: Transfection, Staining, Control, Immunostaining, Knockdown, Comparison, Derivative Assay, Expressing