Figure 6
Rab32 Regulates Some Pluripotent Genes and the Proposed Mechanism of Rab32 during Reprogramming.
(a–f) The expression levels of en-Klf4, en-c-Myc, Klf2, Tbx3, Nr5a2 and Esrrb were determined by qPCR on day 1, 3, 5, 7, 9 and 11 during reprogramming with OSKCtr, OSKR and OSKM. The results of OSKCtr were used as control, *p < 0.05, **p < 0.01. (g) The expression level of Rab32 in G4 cell transduced with two different vectors is increased compared to G4 cells and G4OER cells express substantially higher levels compared to G4OERC cells. (h) The expression levels of en-Klf4, en-c-Myc, Klf2, Tbx3, Nr5a2 and Esrrb were determined by qPCR. The results of G4 were used as control, *p < 0.05; **p < 0.01, ***p < 0.001. Relative expression (g,h) was quantified using the comparative threshold cycle (Ct) method (2–ΔΔCt). The ΔCT was calculated using β-actin, EF1-α and α-tubulin as internal controls. (i) Changes in lipid droplets during reprogramming. The number of lipid droplets per transformed EGFP cell declined sharply during early stages of reprogramming. OSKM cells metabolized most lipids and Rab32 increased the storage of lipids in MEFs, but the lipids level was lower than in OSKCtr. Therefore, OSKR-reprogrammed cells metobolize more lipids than OSKCtr cells. A considerable amount of lipids must be metabolized during early stages of reprogramming and transformed cells began to store lipids at middle stage. (j) After adding Rab32 with OSK, Spot14 was down-expressed, while Mig12, Acaca, Fasn up-regulated, leading to a catalysis forming long-chain fatty acids during early and middle reprogramming stages. The additional expression of Rab32 also led to more expression of c-Myc, Klf2, Tbx3 and Nr5a2. A combination of these results may be the mechanism of improvement in iPSC induction by Rab32.
