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. 2015 Jul 24;10(7):e0133205. doi: 10.1371/journal.pone.0133205

Fig 5. FLASH silencing with siRNAs affects p21 expression in MCs.

Fig 5

Effects of RNAi-mediated silencing of FLASH in cultured mouse MCs. Scrambled siRNA (Scramble) was used as a control. (A) Equal amounts of cell lysates and total RNA were subjected to Western blot (A) and quantative RT-PCR (C), respectively. Since the MCs did not express p16 protein under the usual culture conditions, we have shown tha p16 expression data from p16-expressing MCs induced by cellular responses across multiple passage numbers. PC, positive control (late-passage mouse MCs). One of three independent experiments is shown in (A). (B) FLASH, p21, p27 and p53 protein levels were quantified by densitometric analysis and are expressed as the ratio between these proteins bands optical density and β-actin bands optical density. The quantitative comparison between control siRNA (scrambled) and the most effective siRNA specific for FLASH (RNAi-FLASH3 as KD) were analyzed. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001. (C) Real-time RT-PCR showing relative mRNA levels for FLASH, p16, p21, p27 and p53. The quantitative comparison between scrambled siRNA (Control) and the most effective siRNA specific for FLASH (RNAi-FLASH3 as KD) were analyzed. Results represent as arbitrary units and are shown as mean values ± SDs of at least three independent experiments. NS, not significant. *P<0.001.