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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: Upregulation of lactate-inducible snail protein suppresses oncogene-mediated senescence through p16INK4a inactivation

Fig. 5

Snail mediates the lactate-induced reduction in p16INK4a expression in premalignant cells. a RNA was extracted and subjected to qRT-PCR in B2B cells with indicated lactate treatment. Values represent the relative reduction of p16INK4a mRNA normalized to β-actin. b Indicated cells were transfected with p16INK4a promoters and treated with indicated Lactate for 3 h. Relative luciferase activity is shown. c Scheme of p16INK4aluciferase reporter constructs containing five potential snail binding sites with mutation (1728 M) or not (1728), two potential binding sites (1001) and only one (394). The mutation was done from CAGGTG to TACGGG at the forth potential snail binding site. d Western blot analysis of Snail, p16INK4a and p-Rb in indicated cells at day 3 after infection with Snail siRNA or control and then indicated lactate treatment. e Western blot analysis of Snail, p16INK4a and p-Rb indicated cells at day 3 after infection with Snail cDNA or vector and then indicated lactate treatment. f Indicated cells were transiently co-transfected with Snail cDNA, along with the p16INK4a promoter, and luciferase activity was determined after transfection for 48 h and normalized using the dual luciferase reporter system. g-i Indicated cells were transiently transfected with p16INK4a promoters, along with Snail cDNA or not along with and then with indicated lactate treatment after transfection for 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. j ChIP assays were performed using anti-FLAG antibody. (Left) The Standard PCR products were run and scanned. (Right) The qRT-PCR results were quantified and indicated using the histogram. k, l SA-β-gal activity were analyzed at day 3 after infection with Snail cDNA and p16INK4a, or (L) treatment with different degree of acidification. The percentage of SA-β-gal positive cells is presented in the histogram. The bars represent the mean ± s.d. of triplicates.*P < 0.05, **P < 0.01, ***P < 0.001. m the correlations between SNAI1 and CDKN2A expression from two TCGA data sets (TCGA-LUAD and TCGA-LUSC). The Spearman’s rank correlation coefficient (rho) and the P values were calculated. n Schematic representation of lactate/TGFβ/Snail signaling in the regulation of p16 expression

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