Fig. 3
Depletion of GATA3 in breast cancer cells impairs DNA damage repair in vitro. a Human luminal breast cancer cell line, T47D, was transfected with Si-GATA3-1, Si-GATA3-2, Si-GATA3-3, or Si-control (Si-Ctrl) and analyzed. b–d T47D cells were transfected with Si-GATA3-1, Si-GATA3-2, or Si-Ctrl for 32 h, treated with VP16 at a concentration of 10 μM for additional 0.5 h, allowed to recover for 0 or 20 h, and then immunostained with an antibody against γH2AX and cyclin A (b, c) or 53BP1 (d). Representative γH2AX- and cyclin A-positive cells were shown. Note relative to T47D-Si-Ctrl cells, T47D-Si-GATA3-1 and T47D-Si-GATA3-2 cells retain high levels of γH2AX after 0- and 20-h recovery. γH2AX and cyclin A doubly positive cells are indicated by white arrows. c Quantification of γH2AX-positive cells in b. Only cells with at least 5 γH2AX foci were counted as positive cells. At least 200 cells were counted for each sample. The results represent the mean ± SD of five randomly selected fields per group. The asterisk (*) denotes a statistical significance from Si-Ctrl and Si-GATA3-1 or Si-GATA3-2 samples determined by the T-test. d Quantification of 53BP1-positive cells. Only cells with at least 3 53BP1 foci were counted as positive cells. At least 200 cells were counted for each sample. The results represent the mean ± SD of five randomly selected fields per group. e Analysis of Gata3 levels in MMTV-PyMT (Gata3+/+;Brca1+/+), p18−/−;Gata3+/− (Gata3+/−), and p18−/−;Brca1+/− (Brca1+/−) mouse mammary tumor cells. f, g Gata3+/+;Brca1+/+, Gata3+/−, and Brca1+/− tumor cells were treated with VP16 (f) for 3 h, or E2 (g) for 24 h. h, i The expression of γ-H2AX in f was quantified in h and that in g was quantified in i. j, k T47D-Sh-Ctrl, T47D-Sh-GATA3-1, and T47D-Sh-GATA3-2 cells were treated with E2 for 1 h, and the expression of genes was determined (j) and quantified (k). The blots, images, and graphs represent data from at least three independent experiments