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. 2016 Jan 25;6:19735. doi: 10.1038/srep19735

Figure 2. Study by RNAi to assess the effect of CAR knockdown on HaCaT cell migration and proliferation.

Figure 2

(A) Regimen used in RNAi experiment in vitro. Briefly, HaCaT cells were transfected with either CAR-specific or scramble siRNA duplexes for 24 h. Two days after the completion of RNAi (namely on day 3), western blot, scratch assay, and MTT assay were conducted at selective time points. (B) Representative immunoblots illustrating CAR protein levels at 0 h (day 3) and 60 h (day 5.5) post-RNAi treatment. (C) CAR level in Scramble (Scr) RNAi group after normalization against corresponding β-actin was arbitrarily set at 1. Error bars represent means ± SD from four independent experiments using different batches of cells (n = 4), **p < 0.01. (D) MTT assay was performed on day 3 following RNAi to assess the effects of CAR knockdown on HaCaT cell proliferation at selective time points of 0, 12, 24, 36, and 48 h. Error bars represent means ± SD from four different batches of cells (n = 4), *p < 0.05. (E) Scratch assay illustrating the effects of CAR knockdown on cell migration. On day 3 post-RNAi treatment, HaCaT cells received 1-h mitomycin C treatment, and then subjected to scratch assay. Images were taken at 0, 24, 48, and 60 h post-scratching. Blunted lines indicate the average width of unclosed gap. Scale bar: 100 μm. (F) Scratch gap width at 0 h in each group was arbitrarily set at 1. Error bars represent means ± SD from four independent experiments using different batches of cells (n = 4), **p < 0.01.