Fig. 7

The Notch signaling pathway regulated autophagic activity through NICD-induced phosphorylation of ULK1. A Immunofluorescence staining for NICD (red) and ULK1 (green) in HLE-B3 cells cultured under normal-glucose (NC) and high-glucose conditions (HG), bar = 40 μm. B, C Co-IP of NICD and ULK1 in normal cultured HLE-B3 cells. IP immunoprecipitation, WB Western blot. D Verification of NICD overexpression plasmids, and detection of the levels of autophagy marker proteins and p-ULK1 after transfected with pEGFP-C3 and pEGFP-C3-NICD plasmids for 36 h in normal cultured HLE-B3 cells (NC) by Western blotting. Quantification of the relative levels of NICD (E), p-ULK1/ULK1 (F), LC3II/I G and SQSTM1/p62 (H), n = 3. ns indicates P > 0.05; *P < 0.05, **P < 0.01 and ****P < 0.0001 vs. NC group; ^#P < 0.05, ^##P < 0.01 and ^####P < 0.0001 vs. HG + pEGFP-C3 group. I Verification of ULK1 and ULK1 (Ser 757) overexpression plasmids, and detection of the levels of proteins in the Notch signaling pathway and p-ULK1 after transfected with pcDNA3.1, pcDNA3.1-ULK1, and pcDNA3.1-ULK1 (S758A) plasmids for 36 h, respectively, in HLE-B3 cells cultured in high-glucose conditions (HG) by Western blot assays. Quantification of the relative levels of ULK1 (J), p-ULK1/ULK1 (K), Notch1 (L), NICD M and Snail (N), n = 3. ns indicates P > 0.05; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001 vs. NC group; ^#P < 0.05, ^##P < 0.01 vs. HG + pcDNA group; ^★★P < 0.01 vs. HG + pcDNA-ULK1 group