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. 2021 Jan 8;8:606001. doi: 10.3389/fcell.2020.606001

FIGURE 3.

FIGURE 3

Human epithelial cancer cells sense cytosolic RNA via RIG-I, but not TLR3 or MDA5. (A) PANC-1 cells were transfected with Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT) for 9 h. Cells were then lysed and subjected to western blotting to detect the expression of pPKR, PKR, TLR3, RIG-I, MDA5, LGP2, and DHX29, with β-actin as the loading control. Data are representative of three independent experiments. (B) PANC-1 cells were transfected using Lipofectamine RNAiMAX with non-silencing negative control siRNA (siNC) or siRNA specific for PKR, TLR3, RIG-I, MDA5, LGP2, and DHX29. Forty-eight hours later, RNA was extracted to detect the knockdown level (KD level) by qPCR. (C–E) Silenced PANC-1 cells were transfected with Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT) for 18 h. Culture supernatants were harvested, and IFN-β secretion was measured by ELISA. (F) HCT-8 cells were transfected using Lipofectamine RNAiMAX with non-silencing negative control siRNA (siNC) or siRNA specific for RIG-I to detect the knockdown (KD) level by qPCR. (G–I) Silenced HCT-8 cells were transfected with Poly(I:C)-HMW, Poly(I:C)-LMW, and Poly(dA:dT) for 18 h. Culture supernatants were harvested, and IFN-β secretion was measured by ELISA. All data are shown as mean ± SD of at least three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001).