Pregnancy-Induced Noncoding RNA (PINC) Associates with Polycomb Repressive Complex 2 and Regulates Mammary Epithelial Differentiation
(A, B) To target mPINC1.0 and mPINC1.6 (siPINC1.0/1.6), but not DCR2, siRNAs #1 and #2 were used in combination. To target all splice variants (siPINC), siRNAs #1 and #3 were used in combination. (A) Schematic showing siRNA targets of mPINC splice forms. (B) qPCR shows knockdown of mPINC 5 days post-transfection of siRNAs. Target genes were normalized to Gapdh and set relative to levels in the siNEG control transfected HC11 cells. (C, D) Knockdown of mPNC (siPINC1.0/1.6 and siPINC) splice forms increases Wap and Ltf, but not Csn2, expression at 24 (C) and 72 (D) hrs post-hormone induction. Target genes were normalized to Gapdh and set relative to levels in siNEG treated control cells. Data are presented as mean ± SEM (n = 3). (E, F) Knockdown of mPINC also increases dome formation compared to a control. (E) Representative brightfield images show domes following 48 hrs of hormone treatment. Scale bars represent 50 µm. (F) Domes were counted at 48 hours post-hormone treatment from nine 20× fields/experiment and data represent mean ± SEM set relative to the dome number in the siNEG treated control group (n = 6).