Figure 6.

Small-scaled screening using TMEM192-mKeima system identified TRIM10 and TRIM27 as factors that function in lysophagy. (A) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against each TRIMs or treated with control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM containing Hoechst and observed by fluorescence microscopy. Scale bars, 10 µm. The graph shows lysophagic activity with reference to the wild type. >30 cells were analyzed per condition in each experiment. For lysophagic activity in each siRNA-treated cell, a robust z-score was calculated for the overall lysophagic activity value. Red highlighting indicates those with significantly lower (less than −1.8) z-score values compared with other factors. (B) HeLa cells stably expressing TMEM192-mKeima were treated with siRNA against each of FIP200, TRIM16, TRIM10, TRIM27, or control siRNA. After 48 h of siRNA treatment, cells were cultured for 6 h in the presence of 1 mM LLOMe in DMEM-containing Hoechst and observed by fluorescence microscopy. The graph shows the number of acidic TMEM192-mKeima puncta per cell in each cell. >30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). *P < 0.05; ***P < 0.001 (one-way ANOVA followed by Dunnett’s test). (C) HeLa cells were treated with siRNA against each of TRIM10, TRIM16, TRIM27, or control siRNA. After 48 h of siRNA treatment, cells were treated with or without 1 mM LLOMe for 2 h, immunostained with polyubiquitin chain (FK2) antibody, and then analyzed by fluorescence microscopy. Scale bars, 10 µm. (D) Quantification of the number of polyubiquitin chain (FK2) puncta per cell for Fig. 6 C. More than 30 cells were analyzed per condition in each experiment. Error bars indicate means ± SEM in at least three independent experiments (n = 3). NS: not significant (one-way ANOVA followed by Dunnett’s test).