Figure 3
Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10−8 M E2 up to 72 h. (a) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In (a–f) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. (b) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. (c) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting (d) and MTT assay (e). In (b,d,e), results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels (b) or cell numbers (d,e) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry (f) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry (g). In (f,g), results are the mean ± SEM of three independent experiments. (h & i) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay (h) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB (i) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.
