Figure S3.
CBP and SIRT7 regulate FAM134B-mediated ER fragmentation and ER-phagy. (A and B) CBP KD attenuated FAM134B-mediated ER fragmentation. CBP WT or KD U2OS cells transiently expressing 0.2 µg EGFP-FAM134B (WT) were treated with Tg (1 μM) for 1 h or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in B (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (C and D) CBP WT but not the catalytic activity-dead mutant enhanced FAM134B-mediated ER-phagy. FAM134B KO U2OS cells were engineered to inducibly express mCherry-EGFP-FAM134B (WT) at endogenous levels. CBP was transfected into cells. MCherry-positive but EGFP-negative puncta were quantified in D (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) K160 acetylation of FAM134B in FAM134B-Flag stable cell line was increased by being treated with deacetylase inhibitor NAM (5 mM) for 12 h. IP was performed with an antibody to acetylated lysine. K160 acetylation and Flag were detected by Western blot (WB). (F) K160 acetylation accelerated by SIRT7. Myc-tagged Ctrl, SIRT2, SIRT3, or SIRT7 was expressed individually in FAM134B-Flag stable cell line. The indicated proteins were detected by Western blot. (G and H) SIRT7 KD enhanced FAM134B-mediated ER fragmentation. SIRT7 WT or KD U2OS cells transiently expressing 0.2 µg EGFP-FAM134B (WT) were treated with Tg (1 μM) for 1 h or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in H (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (I) Protein levels in G. SIRT7 WT and KD U2OS cells were treated with 1 μM of Tg for 1 h or DMSO. The indicated proteins were detected by Western blot. (J) Lysosomal cleavage of GFP was analyzed by Western blot in SIRT7 WT or KD cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with Tg (1 μM) for 1 h or DMSO. (K) Lysosomal cleavage of GFP was analyzed by Western blot in SIRT7 WT or KD cells transfected with 1.5 µg GFP-SEC61B and 0.5 µg FAM134B-Flag. Cells were treated with 10 nM BafA1 for 4 h or DMSO. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS3.

CBP and SIRT7 regulate FAM134B-mediated ER fragmentation and ER-phagy. (A and B) CBP KD attenuated FAM134B-mediated ER fragmentation. CBP WT or KD U2OS cells transiently expressing 0.2 µg EGFP-FAM134B (WT) were treated with Tg (1 μM) for 1 h or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in B (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001, ns means no significance. (C and D) CBP WT but not the catalytic activity-dead mutant enhanced FAM134B-mediated ER-phagy. FAM134B KO U2OS cells were engineered to inducibly express mCherry-EGFP-FAM134B (WT) at endogenous levels. CBP was transfected into cells. MCherry-positive but EGFP-negative puncta were quantified in D (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (E) K160 acetylation of FAM134B in FAM134B-Flag stable cell line was increased by being treated with deacetylase inhibitor NAM (5 mM) for 12 h. IP was performed with an antibody to acetylated lysine. K160 acetylation and Flag were detected by Western blot (WB). (F) K160 acetylation accelerated by SIRT7. Myc-tagged Ctrl, SIRT2, SIRT3, or SIRT7 was expressed individually in FAM134B-Flag stable cell line. The indicated proteins were detected by Western blot. (G and H) SIRT7 KD enhanced FAM134B-mediated ER fragmentation. SIRT7 WT or KD U2OS cells transiently expressing 0.2 µg EGFP-FAM134B (WT) were treated with Tg (1 μM) for 1 h or DMSO. Lysosomal degradation of EGFP-FAM134B was blocked by 10 nM BafA1 for 2 h. EGFP-positive puncta were quantified in H (n = 30 cells per group). Scale bars, 10 µm. Data are shown as means ± SEM and analyzed with one-way ANOVA. ***P < 0.001. (I) Protein levels in G. SIRT7 WT and KD U2OS cells were treated with 1 μM of Tg for 1 h or DMSO. The indicated proteins were detected by Western blot. (J) Lysosomal cleavage of GFP was analyzed by Western blot in SIRT7 WT or KD cells transfected with 1.5 μg GFP-SEC61B and 0.5 μg FAM134B-Flag. Cells were treated with Tg (1 μM) for 1 h or DMSO. (K) Lysosomal cleavage of GFP was analyzed by Western blot in SIRT7 WT or KD cells transfected with 1.5 µg GFP-SEC61B and 0.5 µg FAM134B-Flag. Cells were treated with 10 nM BafA1 for 4 h or DMSO. Molecular weight measurements are in kD. Source data are available for this figure: SourceData FS3.

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