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. 2021 Aug 13;12:4928. doi: 10.1038/s41467-021-25145-x

Fig. 8. Bidirectional regulation of LATS2 and autophagy in β-cells.

Fig. 8

a Representative western blot and pooled quantitative densitometry analysis (lower panel) of INS-1E cells transduced with Ad-LacZ or Ad-LATS2 and treated with 50 μM Chloroquine (CQ) for 4 h (n = 3 independent experiments). b Representative western blot and pooled quantitative densitometry analysis (lower panel) of INS-1E cells transfected with siLATS2 or siScr and treated with CQ for 4 h (n = 4 independent experiments). c Representative western blot and pooled quantitative densitometry analysis (lower panel) of human islets transduced with Ad-hShLATS2 or Ad-shScr and treated with Bafilomycin (Baf) or CQ for 4 h (n = 3 different human islets isolations). d Representative western blot and pooled quantitative densitometry analysis (right panel) of INS-1E cells transfected with ATG7 siRNA and/or LATS2 siRNA or control siScr and treated with the 22.2 mM glucose for 24 h (n = 3 independent experiments). e Schematic representation of LysoIP method for immunoprecipitation of intact lysosomes. f INS-1E cells were co-transfected with LATS2-Myc and Tmem192-3xHA or Tmem192-2xFlag plasmids for 48 h. One set of cells were treated with 50 µM CQ for last 4 h. Representative western blot of input and lysosomes isolated from INS-1E cells is shown (n = 2 independent experiments). g Representative western blot and pooled quantitative densitometry analysis (right panel) of INS-1E cells transfected with siScr or siAtg7 for 48 h (n = 3 independent experiments). h Schematic representation of method for immunoprecipitation of autophagosomes. i Stable GFP-LC3 expressing INS-1E cells were transfected with LATS2-Myc plasmid for 48 h. One set of cells were treated with 50 µM CQ for last 4 h. Representative western blot of input and autophagosomes isolated from GFP-LC3 expressing INS-1E cells is shown (n = 2 independent experiments). j GFP-LC3 expressing INS-1E cells were transfected with or without LATS2-Myc plasmid for 48 h. One set of cells were treated with 50 µM CQ for last 4 h. Representative western blot of immunoprecipitation using anti-GFP magnetic beads is shown (n = 2 independent experiments). Data are expressed as means ± SEM. Pooled quantitative densitometry of western blots were normalized to the respective control conditions and ratios, in which a normal distribution of results cannot be proven, were analyzed. *p < 0.05, **p < 0.01; all by two-tailed Student’s t-tests.