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. 2020 Dec 23;18(12):e3001002. doi: 10.1371/journal.pbio.3001002

Fig 2. LSM12 depletion disrupts the RAN gradient and impairs NCT upon oxidative stress.

Fig 2

(A) LSM12 depletion impairs NCT under oxidative stress conditions in a manner that is independent of ATXN2 or SG assembly. Individual shRNA cell lines were transfected with an expression vector for the S-GFP reporter. SG assembly was then quantified 48 hours after transfection. Transfected cells were co-stained with anti-G3BP1 antibody (red) and Hoechst 33258 (blue). Where indicated, cells were incubated with 50-μM NaAsO2 or PBS (vehicle control) for 2 hours to induce oxidative stress. Phospho-EIF2α–dependent SG assembly was blocked by treating with 2-μM ISRIB for 3 hours before NaAsO2 incubation. DMSO was used as vehicle control for ISRIB. (B) NCT of S-GFP reporter proteins was quantified by calculating the ratio of N/C fluorescence in individual cells. Two-way ANOVA detected significant interaction effects of arsenite and ISRIB treatments on NCT only in controlshRNA cells (P = 0.0004). Data represent means ± SEM (n = 100–137 cells from 3 independent experiments). n.s., not significant; ***P < 0.001, as determined by Tukey post hoc test. (C) LSM12 depletion disrupts the nucleocytoplasmic RAN gradient in a manner that is independent of ATXN2 or SG assembly. Cells were incubated with 2-μM ISRIB, 50-μM NaAsO2, or vehicle controls as described above and then co-stained with anti-RAN antibody (red), anti-G3BP1 antibody (green), and Hoechst 33258 (blue). (D) The relative distribution of endogenous RAN proteins was quantified by calculating the ratio of N/C fluorescence. Two-way ANOVA detected significant interaction effects of arsenite and ISRIB treatments on the RAN gradient only in controlshRNA cells (P < 0.0001). Data represent means ± SEM (n = 95–104 cells from 3 independent experiments). n.s., not significant; **P < 0.01, ***P < 0.001, as determined by Tukey post hoc test. All underlying numerical values are available in S1 Data. ANOVA, analysis of variance; ATXN2, ataxin-2; GFP, green fluorescent protein; ISRIB, integrated stress response inhibitor; LSM12, like-Sm protein 12; N/C, nuclear to cytoplasmic; NCT, nucleocytoplasmic transport; SEM, standard error of the mean; SG, stress granule; shRNA, short hairpin.