Figure 3.
Antiapoptotic MCL-1 protein expression and stability are decreased in RPL7a-depleted and ActD-treated Trp53+/+;Eμ-Myc lymphoma cells. (A) Representative western blot analyses of antiapoptotic BCL-2 proteins in control (shRen) and RPL7a- and RPL11-depleted Eμ-Myc lymphoma cells treated as described in Figure 1. Antiapoptotic MCL-1, upper band, is indicated (arrowhead). MCL-1, BCL-2, and BCL-XL protein expression was normalized to α-tubulin and shown as relative to shRen cells (n = 3). (B) Representative western blot analyses of MCL-1 in control (shRen) and RPL7a- and RPL11-depleted Trp53–/–;Eμ-Myc lymphoma cells (n = 3). Levels of antiapoptotic MCL-1 form (arrowhead), normalized to β-actin, are indicated below the corresponding blot as relative to shRen cells. (C) Time-course western blot analyses in Trp53+/+;Eμ-Myc and Trp53–/–;Eμ-Myc lymphoma cells treated with the Pol I inhibitors ActD (5 nM), CX-5461 (50 nM), and BMH-21 (1 μM) for the indicated times (n = 2). (D) Representative western blots showing MCL-1 stability in control (shRen) and RPL7a-depleted Trp53+/+;Eμ-Myc (top panel) or Trp53–/–;Eμ-Myc (bottom panel) lymphoma cells pretreated with doxycycline for 22 hours and harvested after treatment with 100 μg/mL cycloheximide (CHX) at the indicated time points (n = 2-3). Band corresponding to antiapoptotic MCL-1 is indicated (arrowhead). (E-F) Line graphs showing MCL-1 stability over time in control (shRen) and RPL7a-depleted Trp53+/+;Eμ-Myc (E) and Trp53–/–;Eμ-Myc (F) lymphoma cells determined from MCL-1 immunoblots (D) and shown as relative to MCL-1 level at the time of CHX addition. (G-H) Representative western blot and line graph showing MCL-1 stability in Trp53+/+;Eμ-Myc lymphoma cells treated with or without 5 nM ActD for 4 hours (n = 2), determined by CHX chase at the indicated time points, as described in panel E. Band corresponding to antiapoptotic MCL-1 is indicated (arrowhead). NS, not significant. Data are presented as mean ± SEM. *P < .05 (2-way ANOVA test).

Antiapoptotic MCL-1 protein expression and stability are decreased in RPL7a-depleted and ActD-treated Trp53+/+;Eμ-Myc lymphoma cells. (A) Representative western blot analyses of antiapoptotic BCL-2 proteins in control (shRen) and RPL7a- and RPL11-depleted Eμ-Myc lymphoma cells treated as described in Figure 1. Antiapoptotic MCL-1, upper band, is indicated (arrowhead). MCL-1, BCL-2, and BCL-XL protein expression was normalized to α-tubulin and shown as relative to shRen cells (n = 3). (B) Representative western blot analyses of MCL-1 in control (shRen) and RPL7a- and RPL11-depleted Trp53–/–;Eμ-Myc lymphoma cells (n = 3). Levels of antiapoptotic MCL-1 form (arrowhead), normalized to β-actin, are indicated below the corresponding blot as relative to shRen cells. (C) Time-course western blot analyses in Trp53+/+;Eμ-Myc and Trp53–/–;Eμ-Myc lymphoma cells treated with the Pol I inhibitors ActD (5 nM), CX-5461 (50 nM), and BMH-21 (1 μM) for the indicated times (n = 2). (D) Representative western blots showing MCL-1 stability in control (shRen) and RPL7a-depleted Trp53+/+;Eμ-Myc (top panel) or Trp53–/–;Eμ-Myc (bottom panel) lymphoma cells pretreated with doxycycline for 22 hours and harvested after treatment with 100 μg/mL cycloheximide (CHX) at the indicated time points (n = 2-3). Band corresponding to antiapoptotic MCL-1 is indicated (arrowhead). (E-F) Line graphs showing MCL-1 stability over time in control (shRen) and RPL7a-depleted Trp53+/+;Eμ-Myc (E) and Trp53–/–;Eμ-Myc (F) lymphoma cells determined from MCL-1 immunoblots (D) and shown as relative to MCL-1 level at the time of CHX addition. (G-H) Representative western blot and line graph showing MCL-1 stability in Trp53+/+;Eμ-Myc lymphoma cells treated with or without 5 nM ActD for 4 hours (n = 2), determined by CHX chase at the indicated time points, as described in panel E. Band corresponding to antiapoptotic MCL-1 is indicated (arrowhead). NS, not significant. Data are presented as mean ± SEM. *P < .05 (2-way ANOVA test).

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