Fig. 10.
Selective knock-down of LRRC8A abolishes Cisplatin-induced p53, MDM2, and p21Waf1/Cip1 expression and reduces Caspase-9 cleavage in wild-type A549 cells. Western blot analysis of whole cell protein lysates obtained from A549WT, A549CisR10 cells and A549WT cells treated with scramble or LRRC8A siRNA exposed to Cisplatin for 24 h. Protein expression of LRRC8A, p53, p-p53 (Ser15), p21, MDM2, p-MDM2 (Ser166), Noxa, Caspase-9 and Bax, as well as ATM and p-ATM (Ser1981) was determined using specific antibodies (see experimental procedures) and β-actin, histone-3, or α-tubulin served as loading controls (only one is shown). A and B: representative Western blots. C: p53, pp53, p21, Noxa, p-ATM, and MDM2 protein expression relative to their loading controls in A549WT and A549CisR10 (open bars) and following 24 h Cisplatin exposure (black bars). Data represent 3 to 6 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased in Cisplatin-treated cells compared with respective control cells and significantly reduced in Cisplatin-treated A549CisR10 cells compared with Cisplatin-treated A549WT cells, respectively (ANOVA, Fisher LSD method). D: relative protein expression of p53, pp53, p21, Noca, cleaved Caspase-9, and MDM2 in A549WT cell treated with either scramble or LRRC8A siRNA alone (open bars) or in combination with 10 μM Cisplatin (black bars) for 24 h. Data represent 3–6 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased by Cisplatin cell compared with respective control cells and significantly reduced in Cisplatin-treated LRRC8A siRNA cells compared with Scramble siRNA cells, respectively (ANOVA, Fisher LSD method).