Fig. 3

KLF4 enhances 8-Br-cAMP and MPA-induced hESC decidualization by promoting autophagy. A HESCs (from fertile controls, n = 3) were infected with Ad-His-KLF4 or Ad-LacZ (MOI = 25 and 50) for 48 h. LC3-B, LC3-A, Beclin-1, and KLF4 protein levels were analysed by western blot. B hESCs (from fertile controls, n = 3) were infected with Ad-His-KLF4 or Ad-LacZ (MOI = 50) for 24 h followed by treatment with 3-MA (5 mM) for 24 h. LC3-B, LC3-A, Beclin-1, and KLF4 protein levels were analysed by western blot. C and D hESCs (from fertile controls, n = 3) were infected with Ad-His-KLF4 (MOI = 50) or Ad-His-KLF4 (MOI = 50) with 3-MA (5 mM) for 24 h followed by treatment with 0.5 mM 8-Br-cAMP and 1 μM MPA for 3 days. dPRL and IGFBP-1 mRNA levels were measured by qRT–PCR. ^**P < 0.01, ^***P < 0.001. E hESCs (from fertile controls, n = 3) were infected with Ad-LacZ (MOI = 50), Ad-His-KLF4 (MOI = 50) or Ad-His-KLF4 (MOI = 50) with 3-MA (5 mM) for 24 h followed by treatment with 0.5 mM 8-Br-cAMP and 1 μM MPA for 3 days. Prolactin release into the medium was measured by an enzyme-linked fluorescent assay (ELFA). ^*P < 0.05, ^**P < 0.01. F hESCs from fertile controls were infected with Ad-His-KLF4 (MOI = 50) or Ad-His-KLF4 (MOI = 50) with 3-MA (5 mM). After 24 h, the cells were treated with 0.5 mM 8-Br-cAMP and 1 μM MPA as indicated for an additional 72 h. Fluorescein isothiocyanate-labelled phalloidin was used to label actin filaments and to analyse the morphological transformation of hESCs. Scale bar, 50 μm