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. 2018 Jun 13;6(3):277–300. doi: 10.1016/j.jcmgh.2018.06.002

Figure 11.

Figure 11

Validation of gene abrogation by the siRNA approach and validation of the deleterious impact of 5 outlier mutations genes in RKO, KM12, FET, and SW620 CRC cell lines. Gene expression (mRNA level) of outlier mutation–related genes after knock-down by single siRNA was assessed at (A) 24 or (B, left panel) 48 hours after transfection by real-time quantitative PCR. Data represent the means ± SEM of at least 3 independent experiments. (B, right panel) Gene expression (mRNA level) of outlier mutation–related genes after simultaneous down-regulation of 3 genes in HCT116 (upper panel) and SW480 (lower panel) cell lines. (C) Gene expression (mRNA level) of outlier mutation genes after knock-down by siRNA in 4 cell lines (MSI: RKO, KM12; and microsatellite stable: FET, SW620) was assessed 48 hours after transfection by real-time quantitative PCR. Data represent the means ± SEM of 3 independent experiments. (D) Flow cytometry analysis of apoptosis (Annexin V) of untreated (triangle) or TRAIL-treated (circle) MSI (RKO and KM12, left panel) and microsatellite stable (FET and SW620, right panel) CRC cell lines transfected either with a single specific siRNA gene (WNK1, HMGXB4, and/or GART) or with scrambled siRNA. Data represent the means ± SEM of 3 independent experiments. t test: *P < .05, **P < .01 and ***P < .001 of indicated silencing condition compared with control.