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. 2021 Dec 16;22(24):13511. doi: 10.3390/ijms222413511

Figure 2.

Figure 2

Increased basal autophagy in PC1KO mIMCDs. (A) Basal autophagy levels were assessed by Western blotting for LC3 and Sqstm1. Wild-type (WT) and PC1KO mIMCDs were incubated for 24 h in fresh medium and 100 nM Bafilomycin A1 (Baf A1; +) or DMSO (−) was added 3 h prior to harvest. Left: representative Western blot; middle: quantification of LC3-II levels over Actin in absence of Baf A1 (N = 16); right: quantification of LC3-II levels over Actin in presence of Baf A1 (N = 10). (B) Quantification of Sqstm1 levels over Actin in absence of Baf A1 (N = 12). (C) qPCR for LC3 mRNA in WT and PC1KO mIMCDs (N = 6). (D) Representative GFP, DAPI, and merged images of GFP-LC3-transfected WT and PC1KO mIMCDs (scale bar = 20 µm). The insets show the GFP-LC3 punctae. Right: quantification of number of GFP-LC3 punctae per cell. Paired observations of each independent experiment are represented by the same symbol (N = 6). (E) Overexpression of human PC1 (hPC1) in PC1KO restored LC3-II levels. Left: representative Western blot of protein lysates of WT or PC1KO IMCDs transfected with Vehicle (+Veh) or human PC1 (+hPC1); right: quantification of LC3-II over Actin. Paired observations of each independent experiment are represented by the same symbol (N = 5). (F) Analysis of mRFP-GFP-LC3 punctae in transiently transfected WT and PC1KO mIMCDs. Shown are representative GFP, RFP, DAPI and merged images (scale bar = 20 µm) (N = 3). Following addition of Baf A1 (100 nM, 3 h), only combined green and red punctae were observed in WT and PC1KO mIMCDs. (G) qPCR for Atg5, Atg12 and Becn1 mRNA in WT (white) and PC1KO (grey) mIMCDs (N = 3). (H) FoxO1 protein levels in WT and PC1KO mIMCDs. Upper: representative Western blot; lower: quantification of FoxO1 levels over Actin (N = 5). ∗ p < 0.05, ∗∗ p < 0.01.