Figure S2. Translation activity is... | Rockefeller University Press

Figure S2.

$Translation activity is globally suppressed during arsenite stress when VCP or the proteasome are inhibited.(A) U-2 OS cells were unstressed or stressed with 0.5 mM arsenite (As) and/or DMSO (0.1%), DBeQ (10 µM), or MG132 (10 µM) for 30 min. Cells were placed on ice and immediately pelleted and frozen at −80ºC and then lysed and polysome profiles were obtained following ultracentrifugation on sucrose gradients. Shown are representative profiles and fraction RNA in polysome:monosome (P/M) from n = two independent experiments. (B) Metabolic labeling of U-2 OS cells with 35S-Met and 35S-Cys for 30 min was done in the presence or absence of 0.1% DMSO, 10 µM DBeQ, or 10 µM MG132. Cells were lysed, and proteins were run on 4–12% gradient NuPAGE SDS-PAGE gels and exposed to phosphor screens. Total lane intensity was quantified with ImageJ. The average relative percent translation activity from n = two independent replicates is shown ± SEM. Student’s t test was done to assess significance between untreated and treated cells, with ***, P < 0.005. (C) U-2 OS cells were metabolically labeled with 35S-Met and 35S-Cys for 15 min immediately before collection following 0, 15, or 30 min under unstressed or stressed conditions (0.5 mM As) in the presence or absence of DMSO (0.1%), DBeQ (10 µM), or MG132 (10 µM). Translation activity was quantified relative to untreated, unstressed cells as described in B from n = three independent experiments. The average ± SEM is shown for each condition with individual replicates shown in green (As), yellow (As + DMSO), blue (As + DBeQ), and red (As + MG132). Exact P values and source data are provided in Table S1.$

Translation activity is globally suppressed during arsenite stress when VCP or the proteasome are inhibited. (A) U-2 OS cells were unstressed or stressed with 0.5 mM arsenite (As) and/or DMSO (0.1%), DBeQ (10 µM), or MG132 (10 µM) for 30 min. Cells were placed on ice and immediately pelleted and frozen at −80ºC and then lysed and polysome profiles were obtained following ultracentrifugation on sucrose gradients. Shown are representative profiles and fraction RNA in polysome:monosome (P/M) from n = two independent experiments. (B) Metabolic labeling of U-2 OS cells with ³⁵S-Met and ³⁵S-Cys for 30 min was done in the presence or absence of 0.1% DMSO, 10 µM DBeQ, or 10 µM MG132. Cells were lysed, and proteins were run on 4–12% gradient NuPAGE SDS-PAGE gels and exposed to phosphor screens. Total lane intensity was quantified with ImageJ. The average relative percent translation activity from n = two independent replicates is shown ± SEM. Student’s t test was done to assess significance between untreated and treated cells, with ***, P < 0.005. (C) U-2 OS cells were metabolically labeled with ³⁵S-Met and ³⁵S-Cys for 15 min immediately before collection following 0, 15, or 30 min under unstressed or stressed conditions (0.5 mM As) in the presence or absence of DMSO (0.1%), DBeQ (10 µM), or MG132 (10 µM). Translation activity was quantified relative to untreated, unstressed cells as described in B from n = three independent experiments. The average ± SEM is shown for each condition with individual replicates shown in green (As), yellow (As + DMSO), blue (As + DBeQ), and red (As + MG132). Exact P values and source data are provided in Table S1.