Fig. 3

p66Shc knock-down rescues high glucose induced autophagy and senescence of trophoblast cells. A-B qRT-PCR analysis of p66Shc mRNA expression A and Western blot images of p66Shc protein expression (B) in control and p66Shc-KD cells. C qRT-PCR analysis of p66Shc, ATG5 and ATG7 mRNA expressions in control and p66Shc-KD cells. D Western blot images and quantifications of protein levels of ATG5, p62 and LC3 in control and p66Shc-KD cells. E Representative confocal images of autophagic flux in control and p66Shc-KD cells transfected with mCherry-EGFP-LC3 to label the autophagosomes (yellow) and autolysosomes (cherry). Scale bar, 10 μm. F Gene set enrichment analysis of SASP gene signature in control and p66Shc-KD cells. G Western blot images and quantifications of SASP protein expressions of TNF-α, IL-6, IL-1β and p21 in placentae of NP and GDM patients. *P < 0.05, **P < 0.01, ***P < 0.001. H SA-β-gal staining images showing cell senescence in 5 mM or 30 mM glucose treated HTR8/SVneo cells. Scale bar, 100 μm. I Western blot images of p16 and p21 protein levels in 5 mM or 30 mM glucose treated HTR8/SVneo cells. J IHC staining images of p21 in NP and GDM placentae. Green arrowheads indicate upregulation of p21 both in the cytoplasm and nucleus, with a predominant expression in the cytoplasm of syncytiotrophoblast cells in GDM placentae. Scale bar, 100 μm. K SA-β-gal staining showing cell senescence of control and p66Shc-KD cells. L Western blot images and quantifications of protein levels of p16 and p21 in control and p66Shc-KD cells. *P < 0.05, **P < 0.01, ****P < 0.0001