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. 2016 Aug 24;7:12595. doi: 10.1038/ncomms12595

Figure 6. SOX9 is critical for the regulation of breast cancer-initiating capacity downstream PML.

Figure 6

(a,b) Effect of SOX9 silencing with two shRNA (sh9.1 and sh9.2) on SOX9 protein expression (representative western blot out of three independent experiments) (a) and on OSI formation (n=3) (b) in MDA-MB-231 cells. (c) Limiting dilution experiment after xenotransplantation. Nude mice were inoculated with 500,000 or 50,000 MDA-MB-231 cells (n=8 injections per experimental condition). Tumour-initiating cell number was calculated using the ELDA platform. A log-fraction plot of the limiting dilution model fitted to the data is presented. The slope of the line is the log-active cell fraction (solid lines: mean; dotted lines: 95% confidence interval; circles: values obtained in each cell dilution). (d,e) Effect of ectopic SOX9 expression on the consequences of PML silencing. Representative western blot (out of three independent experiments) depicting expression of PML and SOX9 (endogenous and ectopic protein are detected) (d) and OSI formation (n=3) (e) in the different experimental conditions in MDA-MB-231 cells. (f) Limiting dilution experiment to assess frequency of tumour-initiating cells after xenotransplantation. Nude mice were inoculated either with 500,000 or 50,000 MDA-MB-231 cells (n=12 per experimental condition, except in shC/Mock and sh4/SOX9, n=16). Tumour-initiating cell number was calculated using the ELDA platform as in c. (g) OSI formation in MDA-MB-231 cells transduced with the indicated constructs (mock, SOX9) and treated with vehicle or 150 nM ATO (n=6). (h) Diagram of the molecular mechanism by which PML controls the expression of the stem cell factor SOX9 to regulate BCa-initiating cell function. Error bars represent s.e.m., P value (*P<0.05; **P<0.01; ***P<0.001 compared with its control or as indicated). Statistic test: one-tail unpaired t-test (b,e,g) and χ2-test (c,f). ATO, arsenic trioxide; OSI, primary oncospheres; shC, Scramble shRNA; sh9.1 and sh9.2, shRNA against SOX9; sh4, shRNA against PML; VC, vehicle control.