Estrogen Regulation of PCNA Expression

Cells were grown in six-well plates at a density of 5 × 10⁵ cells/mL in 1 % charcoal-stripped FBS media (Invitrogen) for 24 h prior to the start of each experiment. Cells were incubated with vehicle (dimethyl sulfoxide, DMSO, final 0.1 %) or E2 (0, 1, 10, 50, or 100 nM) for 48 h. RNA was extracted from cells using the Promega SV Total RNA Isolation System for Tissues and reverse transcribed (0.2 μg) using the Quanta qScript cDNA supermix (Quanta Biosciences). cDNA was diluted (1:10) and amplified using the Perfecta SYBR Green FastMix (Quanta Biosciences). Groups were normalized to vehicle using the delta delta Ct method (ddCt). Glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA abundance was used to control for DNA template concentrations. Primers for proliferating cell nuclear antigen (PCNA) [17 (link)], forward 5′-CTAGCCATGGGCGTGAAC-3′ reverse 5′-GAATACTAGTGCTAAGGTGTCTGCAT-3′ and GAPDH, forward 5′-GCCAAAAGGGTCATCATCTC-3′ reverse 5′-GGCCATCCACAGTCTTCT-3′ were utilized for this study.