Figure 3: ID3 regulates sensitivity to MEK inhibitors.

(A) Left panel: IC₅₀ analysis of WM4265.2 cells after incubation with siRNA targeting ID3 or non-targeting siRNA for 24 hours; cells were treated with PD901 for 72 hours and cell viability was measured using alamarBlue assays. IC₅₀ values shown are representative of six technical replicates. Middle panel: RT-qPCR of ID3 level, normalized to GAPDH. Values shown are mean +/− standard deviation of n=3. Western blot probed with indicated antibodies of lysates from WM4265.2 cells in the presence or absence of siRNA targeting ID3 and PD901 (10μM). (B) IC₅₀ analysis of WM983B and 1205Lu cells incubated in non-targeting siRNA or siRNA targeting ID3 and treated with PD901 for 72 hours. IC₅₀ values shown represent n=3. (C) IC₅₀ analysis of M93-047 cells with stable knockdown of ID3 via shRNA infection. IC₅₀ values shown represent n=6. (D) Western blot, probed with indicated antibodies of lysates from M93-047 with pooled and two clones of shRNA targeting ID3 or empty vector (EV). (E) Cell cycle analysis of M93-047 cells with stable knockdown of ID3 or empty vector (EV) via flow cytometry. Values shown are the mean +/− standard deviation from n=3. *** p-value < 0.001, * p-value < 0.05; assessed by two tailed student’s t-test. (F) Western blot probed with indicated antibodies of lysates from M93-047 infected with empty vector (EV), shRNA for ID3 (sh1) or shRNA for ID3 plus an ID3 overexpression plasmid that is resistant to silencing (OE). (G) IC₅₀ analysis of the M93-047 clones in F treated with PD901 or Trametinib. IC₅₀ values shown represent n=6.