Figure 4. Identification and validation of the potential importance for CAT-SKL regulation of MBD2.

(A) a time-course microarray approach to measure whole-genome expression identified the methyl-CpG binding domain protein 2 (MBD2) gene as being regulated by CAT-SKL in comparing CAT-SKL-treated and control-treated MDA-MB-468 cell cultures. The time-course experiment collected mRNA data every 6 hours over the course of 30 hours and starting at the zero time-point. (B–C) Mammosphere assay culture conditions were used to test the effect of transient siRNA knockdown of MBD2 expression in FACS-isolated, triple marker-positive MDA-MB-468 CSCs (panel C, 40x magnification). (D) Confirmation of MBD2 knockdown by immunoblot analysis using beta-actin as a control showed that the siRNA treatment was preferentially downregulating a short form variant. The molecular weight corresponds with alternative mRNA splicing variant known to the NCBI as variant 2 (of 2), also known as MBD2c in the literature. For a measure of MBD2a in whole cell lysates the immunoblot exposure time was 15 seconds (s), for MBD2c the exposure time for the same blot was 30 minutes (m). (E) Verification of stable MBD2c overexpression by semiquantitative RT-PCR using RPLPO as the houskeeping control gene (Ct, mean cycle threashold). (F) Verification of stable MBD2c overexpression by immunoblot analysis of nuclear lysates using P62 as a control. All immunoblot bands are cropped, full-length blot images are provided in Supplementary Figure S7. (G,H) Mammosphere assay culture conditions were used to test the effect of stable MBD2c overexpression in MDA-MB-468 cells relative to green fluorescent protein (GFP)-expressing MDA-MB-468 control cells (panel H, 40x magnification). The siRNA knockdown and stable overexpression experiments were done twice, independently and in triplicate, *p < 0.05; ns, not significant.