Fig. 5: Effect of TBK1-FBXO3-TMEM192-TAX1BP1 axis on LLOMe-induced lysophagy.

a HepG2 cells were transfected with both HA-ubiquitin and TMEM192-FLAG and pretreated with either MRT67307 (2 µM) or GSK8612 (10 µM). LLOMe was cotreated for an additional 2 h. Subsequently, the cells were immunoprecipitated with agarose bead-conjugated anti-FLAG antibody and further analyzed by western blotting using the indicated antibodies. Data are presented as the mean ± SEM, with each dot in the plot representing independent triplicate replicates of the experiment (n = 3, *p = 0.0446, **p = 0.0257, ***p = 0.0088). b HepG2 cells overexpressing both GFP-TMEM192 and TAX1BP1-FLAG were pretreated with either BC-1215 or GSK8612. LLOMe was cotreated for an additional 2 h. The cells were then pulled down with an anti-GFP antibody conjugated with agarose beads and further analyzed by western blotting using the indicated antibodies. Data are presented as the mean ± SEM, with each dot in the plot representing independent triplicate replicates of the experiment (n = 3, *p = 0.0171, **p = 0.0006, ***p = 0.0002). c HepG2 cells transiently expressing GFP-LC3 and TMEM192-FLAG were pretreated with either BC-1215 or GSK8612 and cotreated with LLOMe. Subsequently, the cells were stained with an anti-FLAG antibody and imaged by confocal microscopy. The colocalization extent of TMEM192 with LC3 was analyzed using Pearson’s correlation coefficients. Data are presented as the mean ± SEM, with each dot in the plot representing an individual HepG2 cell (n = 15, *p = 0.0031, **p < 0.0001). Scale bar: 10 µm. All experiments were replicated three times. Two-tailed unpaired t-test were used to analyzed all the data. Source data are provided as a Source Data file.