Figure 3: Six1 downregulates p53 independent of MDM2 and induces resistance to MDM2-targeted therapies.

In MCF7-Ctrl and Six1 cells, (a) western blot analysis was performed on whole-cell lysates (WCLs) and nuclear lysates (NLs) 24 h after transfection with an siRNA pool against MDM2 or an siNT. (b) Bright-field images taken 24 h after transfection with an siRNA pool against MDM2. Scale bar, 100 μm (original magnification × 200). (c) Western blot analysis on NL collected 3 h after treatment with Nutlin-3. (d) qRT–PCR in cells collected 4 h after treatment with Nutlin-3. Gene expression is normalized to PPIB. Analysis of variance (ANOVA) on mean±s.d. of biological triplicates for a representative experiment (of >3 experiments). (e) xCELLigence cell proliferation assay. Cells were plated in triplicate to equal confluence and treated with Nutlin-3 (5 μM) and impedance was measured every 30 min for 160 h. The DMSO-normalized slope of the growth curves was calculated over the indicated period of exponential growth. Data shown as mean ±s.d. of triplicate samples for a representative figure (of three experiments). (f) BrdU incorporation assay to measure cell cycle distribution 24 h after treatment with Nutlin-3 (10 μM) or DMSO. The data shown are normalized to the DMSO-treated condition. ANOVA on mean±s.d of biological duplicates from two experiments. (g) T-test on a publicly available gene expression data set of cancer cell lines from multiple tumour types via Oncomine shows that cell lines resistant to Nutlin-3 (IC₅₀>5 μM) have increased expression of Six1. (h) One-tailed χ²-test performed on publicly available patient data sets via cBioPortal^68,69 examining the mutual exclusivity between MDM2 gene amplification and Six1 mRNA overexpression (z-score threshold for Six1 expression is +1.3).