Figure 5
Figure 5. BH3-only proteins Bmf, Noxa, and Bim silencing protected cells from ATO-induced apoptosis. MM.1s and KMS11 cell lines were electroporated with siBmf, siNoxa, siBim, siPuma, and siBid (SmartPool; DHARMACON RNA Technologies). Nontargeting siRNA (si(-)) pool was used as a negative control. After 16 hours, cells were treated with 2 μM ATO for 6 and 24 hours for real-time PCR or Western blot analysis and for 24 and 48 hours for ATO-induced apoptosis analysis. (A) Total RNA was obtained for si(-) control and siBmf samples treated with ATO. Real-time PCR was used to determine Bmf transcript expression. Bmf relative expression refers to Bmf transcript expression relative to GAPDH transcript expression. (B) Protein lysates were obtained for si(-) control–, siNoxa-, siBim-, siPuma-, and siBid-transfected cells and silencing was determined by Western blot at 6 and 24 hours after ATO treatment. (C) siRNA electroporated cells were treated with 2 μM ATO for 24 and 48 hours. Viability was evaluated by annexin-V–FITC/PI staining. Percentage (%) of control (untreated, transfected) viability was plotted versus time (hours). The data are presented as the means (± SD) of 4 and 5 independent experiments for MM.1s and KMS11, respectively. t test was used to compare differences among samples, si(-), and experimental combinations unless otherwise indicated, with confidence intervals of 95%. ND indicates no difference; *P < .05; **P < .01; ***P < .001.

BH3-only proteins Bmf, Noxa, and Bim silencing protected cells from ATO-induced apoptosis. MM.1s and KMS11 cell lines were electroporated with siBmf, siNoxa, siBim, siPuma, and siBid (SmartPool; DHARMACON RNA Technologies). Nontargeting siRNA (si(-)) pool was used as a negative control. After 16 hours, cells were treated with 2 μM ATO for 6 and 24 hours for real-time PCR or Western blot analysis and for 24 and 48 hours for ATO-induced apoptosis analysis. (A) Total RNA was obtained for si(-) control and siBmf samples treated with ATO. Real-time PCR was used to determine Bmf transcript expression. Bmf relative expression refers to Bmf transcript expression relative to GAPDH transcript expression. (B) Protein lysates were obtained for si(-) control–, siNoxa-, siBim-, siPuma-, and siBid-transfected cells and silencing was determined by Western blot at 6 and 24 hours after ATO treatment. (C) siRNA electroporated cells were treated with 2 μM ATO for 24 and 48 hours. Viability was evaluated by annexin-V–FITC/PI staining. Percentage (%) of control (untreated, transfected) viability was plotted versus time (hours). The data are presented as the means (± SD) of 4 and 5 independent experiments for MM.1s and KMS11, respectively. t test was used to compare differences among samples, si(-), and experimental combinations unless otherwise indicated, with confidence intervals of 95%. ND indicates no difference; *P < .05; **P < .01; ***P < .001.

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