Figure 5. PML regulates SOX9 expression in breast cancer.

(a) Flow cytometry analysis of MDA-MB-231 cells based on ALDH1 activity. (b) PML gene expression in the two populations sorted in a (n=3). (c) Expression of self-renewal-associated genes in OSI compared with adherent MDA-MB-231 cells (PML n=6, SOX9 and LGR5 n=5 and SOX2 n=3). (d,e) Representative western blot out of four independent experiments depicting the downregulation of SOX9 protein upon constitutive (d) and inducible (e) PML silencing in MDA-MB-231 cells. (f) Correlation analysis of SOX9 protein densitometry from (e) and OSI formation in MDA-MB-231 cells (n=3). (g) Representative western blot out of four independent experiments depicting the downregulation of SOX9 protein upon 150 nM ATO treatment in MDA-MB-231 cells. (h,i) Representative western blot out of three independent experiments depicting the downregulation of SOX9 protein upon PML silencing (h) and 150 nM ATO treatment (i) in PDX44 cells. (j) PML and SOX9 immunoreactivity assed by immunohistochemistry in a panel of PDX samples (Table 1). (k) SOX9 immunoreactivity in patient biopsies with varying expression of PML in the Marseille cohort (n=737). (l) Cluster score of DNA-binding proteins in SOX9 promoter region using ENCODE database. (m) SOX9 promoter region abundance in chromatin immunoprecipitation (ChIP) of exogenous HA-PMLIV using HA-tag antibody in MDA-MB-231 cells after induction with 50 ng ml⁻¹ doxycycline for 3 days (n=4). Data were normalized to IgG (negative-binding control). Error bars represent s.e.m., P value (*P<0.05, **P<0.01, ***P<0.001 compared with control). Statistic test: one-tail unpaired t-test (b,c,m), Pearson correlation (f), χ²-test (j) and analysis of variance (k). ATO, arsenic trioxide; DEAB, diethylaminobenzaldehyde; dox, doxycycline; OSI, primary oncospheres; shC, Scramble shRNA; sh2, sh4 and sh5, shRNA against PML; VC, vehicle control.