Wisp1 plays a dual role during reprogramming, while Tgfbr2 and Igfbp5 knockdown enhances reprogramming. (A) Potential target genes are efficiently knocked down by siRNAs. Smartpool siRNAs at a final concentration of 50 nM were used to transfect MEFs. Total RNAs were harvested at Day 2 for RT-qPCR to evaluate knockdown efficiency of each siRNA. (B) Knockdown of Tgfbr2 or Igfbp5 enhances Oct4-GFP+ colony formation, while knockdown of Eif4ebp1 and Cxcl14 had no effect. MEFs were transfected with siRNAs on Days 0 and 5 at the same time as 4F infection. GFP+ colonies were counted at Days 11–12 post-infection. Error bars represent three independent experiments with triplicate wells. The P value was calculated using Student's t-test. (**) P < 0.01. (C) Knockdown of Wisp1 shows stage-specific effects on reprogramming. Knockdown of Wisp1 on the same days as 4F transduction (Day 0) decreased the reprogramming efficiency by ∼70%, while knockdown on Day 5 enhanced reprogramming by approximately threefold. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. (D) Wisp1 is efficiently knocked down by siRNAs during both procedures. siWisp1 was transfected at a final concentration of 50 nM on Day 0 or Day 5. Total RNAs were harvested at Day 2 post-transfection for RT-qPCR analysis of Wisp1 expression. (E) Knockdown of Wisp1 is able to rescue iPS reprogramming after inhibition of miR-135b at Day 5. Error bars represent three independent experiments with duplicate wells. (F) Knockdown of Wisp1 at Day 0 inhibits mesenchymal-to-epithelial transition (MET). MEFs were infected with 4F and transfected with siRNA on the same day (Day 0). Total RNAs were harvested 2 d later. Expression of several MET markers was evaluated. (G) Knockdown of Wisp1 at Day 5 does not affect MET. MEFs were transduced with 4F at Day 0 and transfected with siRNA at Day 5 post-4F infection. Total RNAs were harvested 2 d after transfection and expression of the MET markers was evaluated. (H) Overexpression of Wisp1 inhibits iPS induction. MEFs were transduced with a Wisp1 HA tagged retroviral vector along with OSKM, and GFP colonies were quantified on Days 12–14. RT-qPCR data were analyzed using the Wilcoxon rank-sum test. (*) P < 0.05; (**) P < 0.01.
