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. 2023 May 15;12(1):26. doi: 10.1038/s41389-023-00473-3

Fig. 4. ANGPTL8 activates ERK pathway-mediated autophagy and promotes the proliferation of HCC cells.

Fig. 4

A, B Beclin-1, ATG5, P62, and LC3II/I levels in ANGPTL8-overexpressing A MHCC97H and B HepG2 cells treated with or without the ERK inhibitor FR180204 were detected by western blotting. Protein expression was normalized to β-tubulin or GAPDH, and the numbers represent the mean ± SD of the average of 3 independent experiments. C, D Beclin-1, ATG5, P62, and LC3II/I levels in C WT and D ANGPTL8-KO primary mouse hepatocytes cultured with or without rANGPTL8 and FR180204 were detected by western blotting. Protein expression was normalized to β-tubulin or GAPDH, and the numbers represent the mean ± SD of an average of 3 independent experiments. E (a) Autophagy levels in primary mouse hepatocytes treated with rANGPTL8 or the ERK inhibitor FR180204 were monitored using the fluorescence reporter Ad-GFP-LC3B, and (b) autophagy levels were quantified by calculating the number of green dots (n = 8 per group). Data are the mean ± SD. Statistical comparisons were performed using Student’s t test. ****p < 0.0001. F (a) Autophagic flux in primary mouse hepatocytes treated with rANGPTL8 or the ERK inhibitor FR180204 was monitored using the fluorescence reporter Adplus-mCherry-GFP-LC3B. (b) Autophagy levels were quantified by calculating the number of red and yellow puncta (n = 3 per group). Data are the mean ± SD. Statistical comparisons were performed using Student’s t test. *p < 0.05, **p < 0.01. G Phosphorylated ERK (P-ERK), ERK, ATG5 and LC3II/I in ANGPTL8-OE (a) MHCC97H and (b) HepG2 cells with or without the autophagy inhibitor CQ (50 μΜ, 24 h) were detected by western blotting. H Phosphorylated ERK (P-ERK), ERK, ATG5 and LC3II/I in primary mouse hepatocytes from (a) WT and (b) ANGPTL8-KO mice with or without rANGPTL8 and CQ were detected by western blotting. I The proliferation of ANGPTL8-overexpressing MHCC97H, HepG2, and LO2 cells treated with or without CQ was monitored by RTCA.