Fig. 4

Knockdowns of CPSF3 or hnRNP UL1 impair MEF reprogramming. A Gene expression profiles of Cpsf3 in B cell reprogramming and MEF reprogramming (cpm values, blue and magenta lines, respectively). The x-axis represents “reprogramming pseudotime” in both datasets, calculated through the PCA analysis of Figure 2B. B Gene expression levels of Hnrnpul1 in B cell and MEF reprogramming (cpm values, blue and magenta lines respectively), as in panel A. C Relative expression levels of Cpsf3 mRNA quantified by RT-qPCR in non-infected cells (NI), cells transduced with a scrambled control shRNA (shSCR) or one of two shRNAs specific for Cpsf3. The y-axis represents the relative expression (2^(−∆Ct) value) of Cpsf3 after normalization over Gapdh. D Relative expression levels of Hnrnpul1 mRNA quantified by RT-qPCR as in panel C. E Relative expression levels of Pou5f1 and Nanog quantified by RT-qPCR as in panels C and D. See also Additional file 1: Figure S4B. B–E Average of biological triplicates and SD values are shown. Statistical significance was calculated by t-test on ∆Ct values compared to the NI control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing using Holm-Sidak method). F Reduction of early reprogramming intermediates at day 6 post-OSKM induction upon knockdowns of Cpsf3 and Hnrnpul1. Fold change was calculated from the percentage of SSEA1+EPCAM1− cells (of the total of alive cells) in every condition compared to the shSCR control using flow cytometry analysis. G Reduction of late reprogramming intermediates at days 10 and 12 post-OSKM induction upon knockdown of Cpsf3 or Hnrnpul1. Fold change was calculated from the percentage of SSEA1+EPCAM1+ cells (of the total of alive cells) in every condition compared to the shSCR control using flow cytometry analysis. See Additional file 1: Figure S4C for examples of gates. H Number of colonies stained with alkaline phosphatase (AP) at day 14 post-OSKM induction upon knockdown of Cpsf3 or Hnrnpul1. On the bottom, images of representative wells are shown for every condition. F,G,H Average of biological triplicates and SD values are shown. Statistical significance was calculated by t-test comparing each condition to the shSCR control (*, **, *** = p value < 0.05, 0.01, 0.001 respectively, corrected for multiple testing with Holm-Sidak method)