Fig. 4

Loss of Ate1 impairs p62 body clearance. WT, Ate1 KO, and Ate1 KO^Ate1-1 cells were cultured in serum-rich (CONTROL) conditions or incubated with 20 μM CQ under STV conditions (STV/CQ) for 6 h. a Immunostaining of p62 bodies was performed using p62 (red hot), Ate1 (green), and poly-Ub (pUb, magenta) antibodies and visualized with confocal microscopy. Images are maximum intensity projections of confocal “z” stacks, and insets show single plane images at higher magnification of the selected regions (white dashed box). b Quantification of the size (upper panel) and number (lower panel) of p62 bodies, relative to cell area. The relative area covered by and the number of p62-labeled inclusion bodies was quantified using the “Analyze particles” tool of Fiji ImageJ software. CONTROL, n=242/337/50; STV/CQ, n=112/132/138 bodies were analyzed in WT, Ate1 KO, and Ate1 KO^Ate1-1 cells, respectively. c Immunoblot analysis of p62 protein levels. Ate1 and Gapdh were used as Ate1 deletion and loading controls, respectively. (*) p62 antibody IgG heavy chain. Densitometric analysis was performed, and the relative p62 expression levels are provided below each lane. d Quantification of poly-Ub puncta fluorescence intensity. The relative area covered by poly-Ub labeled puncta was quantified using the “Analyze particles” tool of Fiji ImageJ software. CONTROL, n=3702/2446/622; STV/CQ, n=3768/1360/1377 puncta were analyzed in WT, Ate1 KO, and Ate1 KO^Ate1-1 cells, respectively. Statistical significance was assessed using a two-sided unpaired t test. ****P≤0.0001, ns=nonsignificant. Scale bars: 10 μm (main images), 3 μm (inset)