Validating Knockdown Efficiency of shRNAs

Short hairpin shRNA (shRNA) vectors for human LRRC8A, TTYH1, and TTYH2 were constructed using pSicoR (Addgene, #11579) with GFP replaced by mCherry. shRNA sequences were as follows: LRRC8A: 5′-GGTACAACCACATCGCCTA-3′ [3 (link)]; TTYH1: 5′-GCATCGGTTTCTATGGCAACA-3′; TTYH2: 5′-GGATTATCTGGACGCTCTTGC-3′ [13 (link)]. A pSicoR construct containing a scrambled shRNA was used as a control. To validate shRNAs, SNU-601, HEK293T, HepG2, and LoVo cells were cultured and transfected with each shRNA in the presence of Lipofectamine 2000 (Invitrogen) in serum-free culture medium for 6 h, and then incubated in normal culture medium for 72 h. The efficiency of gene silencing was assessed by RT-PCR and western blot.