Figure 5: EpEX/EpCAM-mediated iPSC reprogramming through STAT3 signal.

(A) The phospho-kinase array detects phosphorylated proteins in untreated and EpEX-treated MEFs. (B) Quantification by mean pixel density revealed that six phosphorylated proteins are regulated by EpEX. (C) MEFs are stimulated by EpEX in a time-dependent manner. MEFs were treated by EpEX (1 μg/mL) for 5, 15, 30, 60, or 120 min. After treatment, cells were harvested, and phosphorylation of STAT proteins was detected by Western blotting (n = 3). (D,E) MEFs were stimulated by EpEX in dose- and time-dependent manners. MEFs were treated with EpEX at concentrations of 0, 0.5, 1, 2.5, 5, 10 μg/mL for 15 min, or with EpEX (1 μg/mL) for 5, 15, 30, 60, 120, 240, 360 min. After treatment, cells were harvested, and the phosphorylation of STAT3 was detected by Western blotting (n = 3). (F) MEFs were stimulated with EpEX (1 μg/mL) and/or LIF (1 μM) for 1 h. The phosphorylation of STAT3 (Tyr 705) was examined by Western blotting (n = 3). (G and H) MEFs were transfected with OSKM, OE, and KE, and treated with EpEX. After transfection, cells in each group (including control MEFs) were stimulated with doxycycline (1.5 μg/mL) in iPSC medium (DMEM/F12 with stem cell knockout serum, without LIF) and harvested on days 3, 6, or 9. STAT3 and phopho-STAT3 protein expression was detected by Western blotting (n = 3). Kinetic expression was shown after quantification.