ON-TARGETplus Human LRRC8A (56262) siRNA - SMARTpool

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	Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

Protocol tips

Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

Publication protocol

Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

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