Fig 2: Resveratrol-activated SirT1 deacetylates Snail and increases Snail stability. a Western blot demonstrates increased Snail expression following 3 h of resveratrol (0, 1, 5, 10, 20 and 30 µM) stimulation in different histological non-small-cell lung cancer cell lines. b RNA was extracted and subjected to qRT-PCR in A549 and H1299 cells with indicated resveratrol treatment. Values represent the relative reduction of Snail mRNA levels normalized to GAPDH. No significant differences were observed (difference from untreated cells by ANOVA with Dunnett’s correction for multiple comparisons was calculated). c A549 and H1299 cells were co-transfected with Snail promoter reporter and control Renilla luciferase reporter gene plasmid for 48 h and then treated with resveratrol for 3 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. No significant differences were found. d The half-life of Snail was increased in cells treated with resveratrol compared with that in control cells, as demonstrated by CHX chase assays. Cells were exposed to CHX and collected at the indicated times. Cell lysates were immunoblotted with the indicated antibodies. e Western blot analysis of Snail in H1299 and A549 cells at day 3 after transfection with SirT1 cDNA or control vector and with resveratrol treatment. f Western blot analysis of Snail in the cells with resveratrol treatment at day 3 after transfection with wild-type SirT1 cDNA (SirT1/wt) or acetylase-dead SirT1 construct (SirT1/Mut) or empty vector. g Western blot analysis of Snail in the cells at day 3 after transfection with SirT1 siRNA or control siRNA. h Cells were transfected with SirT1 for 48 h, and then cell homogenates were immunoprecipitated with anti-Snail and blotted with anti-acetyl-lysine. i Co-immunoprecipitation assays were used to detect the direct interaction of SirT1 and Snail after transfection with SirT1 in the H1299 and A549 cells

Fig 3: HIF-1a is a key regulator of drug-induced metabolic reprogramming. a and b Western blot demonstrated the time-course (a) and dose-dependent (b) induction of HIF1-a by etoposide. c A549 and H1299 cells were transfected with a HRE-luc plasmid containing five synthetic responsive elements of HIF1-a for 24 h and then treated with etoposide for 5 h. Then luciferase activity was immediately measured and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (*p < 0.05 for difference from untreated cells by ANOVA for multiple comparison). d A549 Cells were treated with etoposide for 5 h before determination of ROS. e A549 and H1299 cells were pretreated with increasing concentrations of N-acetylcysteine (NAC) for 30 min before 25 µM etoposide was added. Western blot was conducted 5 h later to determine levels of HIF-1a, HK2, MCT4, Glut1. f A549 cells were transfected with HIF-1a siRNA for 24 h and further treated with 25 µM etoposide for 36 h. Western blot was performed to examine the expression of HK2, MCT4, Glut1. g The cells were first stimulated either with different concentrations of LA or different degree of acidification by adding HCl into medium for 3 h followed by further treatment with 25 µM etoposide for additional 36 h and then lysed for western blot analysis of MRP1, cleaved-PARP and Snail expression. h Gradual titration of NaHCO3 to LA-rich medium dramatically sensitize A549 cells to etoposide treatment as indicated by Cleaved-PARP levels. i Cell apoptosis assay. Addition of NaHCO3 rescued the etoposide-induced apoptosis inhibited by lactate treatment by Annexin-PI dual staining. The quantification was presented in right panel (**p < 0.01, ***p < 0.001, for difference from the untreated cells, ##p<0.01 for difference from etoposide-transfected cells, $$p < 0.01 for difference from the combined etoposide and LA treated cells by ANOVA with Dunnett’s correction for multiple comparisons) j Cell viability assay showed titration of lactic acid with NaNCO3 rescued the etoposide-induced inhibition of cell viability. Corresponding EC50 values for etoposide, Etoposide+LA and Etoposide+LA + NaNCO3 are 20.11 µM, 27.99 µM and 19.71 µM respectively

Fig 4: eHsp90 enhances ALDH activity via Snail and EZH2A, B. Top panels show representative flow cytometry scatter plots for the ALDEFLUOR assay of ARCaPE-LacZ relative to ARCaPE-eHsp90 (A) and DU145-LacZ relative to DU145-eHsp90 (B). Middle panels show representative scatter plots for ARCaPE-eHsp90 and DU145-eHsp90 transduced with either an shGFP vector control, shSnail, or shEZH2. Lower panels show representative scatter plots for ARCaPE-eHsp90 and DU145-eHsp90 treated with 100 nM SCH or 500 nM GSK343 over a 3 day period. Comparisons included eHsp90-expressing cells relative to the matched LacZ control. As indicated, additional treatments or cell derivatives were compared relative to the matched untreated eHsp90-expressing cells. Combined analysis of replicate assays are shown in accompanying graphs. All statistics were performed using the Student's t-test. * = p<0.05, ** p<0.01.

Fig 5: Ectopic expression of Snail in Madin-Darby bovine kidney (MDBK) cells does not induce DNA methylation of the E-cadherin promoter. Diagram showing the position of 4 E-boxes (-403 to -398, -201 to -196, -151 to -146, and -100 to -95; red bars) and CpG dinucleotides within the E-cadherin promoter region (circles). Genomic DNA was isolated from the control cells [transfected with a neomycin resistance gene (neo) cells] and Snail cells (cells ectopically expressing Snail protein), and the methylation of the E-cadherin promoter was analyzed by bisulfite sequencing. Genomic DNA incubated with CpG methyl-transfease prior to bisulfite treatment was used as a positive control for methylated DNA. Methylated and unmethylated dinucleotides are indicated as filled and open circles, respectively.