Skip to main content
. Author manuscript; available in PMC: 2016 Jul 11.
Published in final edited form as: Cell Rep. 2016 Jun 2;15(11):2488–2499. doi: 10.1016/j.celrep.2016.05.031

Figure 3. BRCA1 and BRCA2-deficient tumor cells are hyperdependent on monoubiquitinated FANCD2 for survival.

Figure 3

(A) Clonogenic formation of a panel of 18 breast cell lines transfected with Scr or FANCD2 siRNA. Percent survival of cells transfected with siFANCD2 versus siScr is plotted. The genetic status of the cell line analyzed is indicated (Nle: normal, non-transformed mammary epithelial cell line; TNBC: triple negative breast cancer; HRP: HR-proficient; HRD: HR-deficient). FANCD2 gene expression in the 58 breast cell lines from the CCLE collection that includes 16 of the 18 breast cell lines tested in Figure 3A. Cell lines are grouped based on their genetic status. For each group, expression values are represented as the mean of z-scores. (Hor. pos.: hormone positive breast cancer). A nonparametric test (Mann-Withney-Wilcoxon) was used to compare the value of the HRD TNBC group with the rest of the groups. (B) Growth of MDA-MB-436 cells expressing doxycycline (dox)-inducible FANCD2 or scrambled (Scr) shRNA in athymic nude mice. Immunoblot showing silencing efficiency. (C) Relative tumour volumes (RTV) for individual mice treated in (B) after five weeks of doxycycline (dox) treatment. Each group represents n≥10 tumors from n≥6 mice while each dot represents data from one tumor. Data are shown as mean ± s.e.m. Student’s t test was used to compare each sample to “shFANCD2; dox”. (D) Clonogenic formation of FANCD2-deficient (PD20) cells expressing empty vector (EV) or FANCD2 cDNA constructs and transfected with BRCA2 siRNA. Percent survival of cells transfected with BRCA2 versus Scr siRNA is plotted. An Immunoblot shows expression of FANCD2 cDNA constructs. Representative images are shown. (E) Clonogenic formation of FANCD2-deficient (PD20) cells expressing EV or FANCD2 cDNA constructs and transfected with BRCA2 siRNA in increasing concentration of MMC. Percent survival relative to non-treated cells (MMC 0 ng/mL) is plotted. Data in A, D–E represent mean ± s.e.m. over n=3 independent experiments. Data in D was analyzed using the Chi-squared test for trend in proportions. Abbreviations: ns, non significant; EV, empty vector; WT, wild-type.