Figure 1.

CRISPR/Cas9-mediated SIRT6 knockout inhibits growth and clonogenic survival, induces G1-phase arrest in human melanoma cells. A375 SIRT6 WT and KO clones were seeded and analyzed after 72 h, with different assays. (A) Cell lysates (with equal amount of protein) were subjected to immunoblot analysis to confirm SIRT6 KO. β-tubulin was used as a loading control. (B) Cell growth and viability were determined by trypan blue exclusion assay. Results are expressed as percentage of viable or total SIRT6 KO cells compared to WT. (C) To determine the clonogenic survival, an equal number of viable cells were plated into 6-well plates at low density. After ∼10 days, cells were fixed and stained with crystal violet. The images shown are representative of three experiments with similar results. (D) Cell cycle analysis was performed using propidium iodide (PI) staining, and data were analyzed with ModFit software, representative histograms are shown. (E) Mean percent of cells in each phase of the cell cycle are shown. The data are expressed as mean ±SEM of three experiments, done in triplicate. Statistical significance is determined by two-way ANOVA analyzed via GraphPad Prism Software and is denoted as **P≤0.01, ****P≤0.0001.