Fig. 3 | Journal of Cancer Research and Clinical Oncology

Fig. 3

From: Resveratrol induces PD-L1 expression through snail-driven activation of Wnt pathway in lung cancer cells

Fig. 3

Resveratrol induces Snail-dependent reduction of Axin2 levels. a Western blot analysis of Axin2 and PD-L1 in H1299 and A549 cells at day 3 after transfection with Axin2 cDNA or vector and then treated with resveratrol for 3 h. b RNA was extracted and subjected to qRT-PCR in A549 and H1299 cells with indicated concentrations of resveratrol treatment. Values represent the relative reduction of Axin2 mRNA levels normalized to GAPDH. The bars represent the mean ± SD of triplicates (**p < 0.01, ****p < 0.0001 for difference from control cells by ANOVA with Dunnett’s correction for multiple comparisons). c A549 and H1299 cells were co-transfected with Axin2 promoter reporter construct and control Renilla luciferase reporter gene plasmid and treated with indicated resveratrol concentrations after 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (**p < 0.01, ***p < 0.001, ****p < 0.001 for difference from control cells by ANOVA with Dunnett’s correction for multiple comparisons). d Schematic representation of potential Snail binding sites in the Axin2 promoter. The mutation construct (917M) was done from CAGGTG to AGTCAC at the potential Snail binding site. e Western blot examines epithelial marker E-cadherin, mesenchymal markers N-cadherin, Fibronectin, Vimentin, and EMT inducer Snail in H1299 and A549 cells with different doses of resveratrol treatment. f A549 and H1299 cells were transiently co-transfected with Snail cDNA, along with the Axin2 promoter reporter, and luciferase activity was determined and normalized after transfection for 48 h using the dual luciferase reporter system (*p < 0.05, ***p < 0.001, for difference from vector-transfected cells by ANOVA with Dunnett’s correction for multiple comparisons). g The cells were transiently transfected with wild-type and mutated Axin2 promoter construct for 48 h and treated with resveratrol for 3 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (****p < 0.001 for difference from untreated transfected-cells by ANOVA with Dunnett’s correction for multiple comparisons). h ChIP assays were performed using anti-FLAG antibodies. The standard PCR product is run and scan (left panel). Quantitative RT-PCR results were expressed using histograms (right panel). The bars represent the mean ± SD of triplicates (****p < 0.0001 for difference from empty control plasmid-transfected cells by ANOVA for multiple comparison)

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