Fig. 6: Association of FBXO3 with LLOMe-induced lysophagy in FBXO3 knockout HepG2 cells.

a FBXO3 wild-type (WT) and knockout (KO) HepG2 cells were analyzed using western blotting with an FBXO3 antibody. Data are presented as the mean (n = 2). b FBXO3 WT and KO HepG2 cells expressing GFP-Gal3 were treated with LLOMe (2 mM, 3 h), washed, and incubated with fresh medium. The number of GFP-Gal3 puncta per cell was counted. Data are presented as the mean ± SEM, with each dot representing an individual HepG2 cell (n = 470, *p < 0.0001). Scale bar: 10 µm. c FBXO3 WT and KO HepG2 cells expressing HA-ubiquitin and TMEM192-FLAG were treated with LLOMe. The cells were immunoprecipitated with anti-TMEM192 antibody and analyzed by western blotting. Data are presented as the mean ± SEM (n = 3, *p = 0.0174, **p = 0.0011). d FBXO3 WT and KO HepG2 cells expressing EGFP-TMEM192 and TAX1BP1-FLAG were treated with LLOMe. Then, the cells were immunoprecipitated with anti-EGFP antibody conjugated with agarose beads and analyzed by western blotting. Data are presented as the mean (n = 2, *p = 0.0355, **p = 0.0066). e FBXO3 WT and KO HepG2 cells transiently expressing mCherry-Gal3 were reconstituted with empty EGFP, EGFP-FBXO3 WT, or EGFP-FBXO3 V221I. The cells were treated with LLOMe, washed, and incubated with fresh culture medium for 24 h. The number of mCherry-Gal3 puncta per cell was counted. Data are presented as the mean ± SEM, with each dot representing an individual HepG2 cell (n = 200, *p < 0.0001). Scale bar: 10 µm. f FBXO3 WT and KO HepG2 cells expressing HA-ubiquitin in combination with empty EGFP, EGFP-FBXO3 WT, or V221I were treated with LLOMe. The cells were then immunoprecipitated with anti-TMEM192 antibody and analyzed by western blotting. Data are presented as the mean ± SEM (n = 3, *p = 0.0161, **p = 0.0039). All experiments were replicated three times except d (twice replicates). Each dot in the plot a, c, d, and f represents independent replicates of the experiment. Two-tailed unpaired t-test were used to analyzed all the data. Source data are provided as a Source Data file.