Figure 7. γKlotho protects cancer cells against ROS toxicity.

A. γKlotho overexpression reduces ROS levels in unstressed cells. Cells overexpressing control vector or γKlotho-Flag were seeded in 6 well plates, and harvested the next day. Before harvest, 10 μM DCF-DA was added to the cells, cells were incubated for 45 minutes at 37°C, then washed with PBS, trypsinized, and stained with PI before FACS analysis. Representative histograms are shown. B. Quantification of ROS positive live HS578T and MDA-MB-231 cells overexpressing control vector or γKlotho-Flag. Data are mean ± SD of 2 replicates. p ≤ 0.05, **p ≤ 0.01; t-test C. Control vector or γKlotho-Flag overexpressing cells were treated with 10 μM or 100 μM H₂O₂ when 10 μM DCF-DA was added to the cells. Staining and FACS analysis was performed as in A. Representative histograms are shown. D. Quantification of ROS positive live cells. Data are mean ± SD of 2 replicates. E. HS578T cells stably expressing vector or γKlotho-Flag were seeded in 96-well plates at 6500 cells/well. 12 hours later, culture media was replaced with increasing concentration of H₂O₂ in serum free RPMI media. 72 hours later MTS viability assay was performed. Data are normalized to the untreated controls (100% viability). Dose response curves are plotted using a non-linear regression model and IC₅₀s were determined from the fitted curves using Hill equation. F. 100,000 HS578T cells expressing control vector or γKlotho were seeded in 12-well plates, next day treated with the indicated concentrations of H₂O₂ in serum free RPMI for 1 h at 37°C and then re-plated into 12-well plates at low density (2500 cells/well). Two weeks after plating, cells were fixed in ice-cold methanol and stained with crystal violet. Representative images of 2 independent experiments are shown. Cell growth was quantified by determining the percentage of area covered by crystal violet stained cell colonies. Data are presented as mean of three replicates ± SD from one of the experiments. *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001; two way ANOVA. G. MDA-MB-231 cells stably expressing control vector or γKlotho-FLAG were seeded in 96-well plates at 8000 cells/well and cell viability after H₂0₂-induced oxidative stress measured as in E. H. 100,000 MDA-MB-231 cells expressing control vector or γKlotho were treated as in F, then re-plated in 12-well plates at 5000 cells/well. Cell growth was quantified and is presented as in F. *p ≤ 0.05, ***p ≤ 0.001; two way ANOVA. I. HS578T-vector and γKlotho-Flag cells were treated with increasing concentration of doxorubicin as in E. J. The working model of γKlotho role in triple negative breast cancer cells.