Supplementary figure S2 from Chemical Inhibition of Wild-Type p53-Induced Phosphatase 1 (WIP1/PPM1D) by GSK2830371 Potentiates the Sensitivity to MDM2 Inhibitors in a p53-Dependent Manner

Supplementary figure S2: A) Western blot shows optimisation of anti-WIP1 antibody (F-10) using siRNA mediated knockdown in MCF-7 cells. Following 24 and 48 hours of siRNA mediated knockdown conditions, MCF-7 cells were treated with 10Gy IR, lysates were collected after 4 hours and analysed by western blotting. All the bands shown were knocked down in response to four different WIP1 targeting siRNA constructs (WIP1.1-4) compared to the universal control siRNA (Cont. siRNA). WIP1 knockdown also resulted in higher and persistent pp53Ser15 most notably in response to the WIP1.2 siRNA construct. WIP1.1 Sense: GUGCCAUAGUAAUCUGCAU; WIP1.2 Sense: GGUGUAGUCAUACCCUCAA; WIP1.3 Sense: GCCCUUCCUAUAAUAGUCA; WIP1.4 Sense: UUGGCCUUGUGCCUACUAA Universal control Sense: GCGCGCUUUGUAGGAUUCG. B) Nutlin-3 and RG7388 SRB growth inhibition curves in MCF-7 cells in the presence and absence of 2.5µM GSK2830371. C) On the left side two independent repeats of the western blot in Figure 2A showing degradation of WIP1 protein in MCF-7 cells over time following treatment with 2.5µM GSK2830371. Full-length WIP1 band optical density (OD) was normalised to actin OD over time.