Figure 1.

USP9X depletion reduces ReNcells VM proliferation. (A) Doxycycline treatment depleted USP9X protein levels after 24, 36 and 72 hours in ReNcell VM cells harbouring USP9X-targeted shRNA (2913 and 4774) but not a nonsense shRNA (Scrambled) or non-transduced cells (Wildtype). Blot representative of six biological replicates. Gels were run under the same experimental conditions. USP9X and β-tubulin proteins at expected molecular weights. (B) Reduced Cell index of ReNcell VM cells following addition of 1 μM Doxycycline (0 hour). xCELLigence real-time measurement of increasing electrical impedance plotted on “Y axis” (“Normalised Cell Index”). Cell Index of doxycycline treated (+) ‘2193’ ReNCell VM cells differed significantly from non-treated after 34 hours (p < 0.05), 35 hours (p < 0.01) and 36 hours (p < 0.001). After 72 hours, the cell index was approximately 50% of untreated 2193 cells. For ‘4774’ cells, the cell index differed significantly at 28 hours (p < 0.05), 29 hours (p < 0.01) and 30 hours (p < 0.001) after treatment. After 72 hrs, the reduction in cell index was approximately 61% that of untreated 4774 cells. Analysis was obtained by plotting t-values of the two way ANOVA analysis (versus time). Error bars represent standard deviation. wt: wildtype, scr: scrambled, +: 1 μM doxycycline treated, −: No doxycycline. Representative of two biological replicates. (C) ReNcell VM cells exposed to doxycycline for 72 hours were labelled with a 6 hour pulse of EdU, to mark cells undergoing DNA-synthesis in S-phase, before immunofluorescence staining for GFP (transduced cells) and EdU (Red). (D) Quantitation of cell counts revealed significantly fewer EdU-positive cells in doxycycline-treated 2193 and 4774 cells compared to Scrambled. A minimum of 200 cells from each of two biological replicates, for each treatment, was counted. All data are means ± SEM. Statistical significance was assessed by one-way ANOVA, followed by Tukey’s post-test. **p < 0.01.