Fig. 1

Proof of concept for PcG/trxG-mediated regulation of PRE-EGFP and PRE-F.Luc reporters. a Drosophila Schneider (S2) cells co-transfected with PRE-EGFP and pActin-RFP show high levels of EGFP and RFP expression. Co-transfection of PRE–EGFP + pActin-RFP reporters with Pc b or E(z) c over-expressing constructs resulted in strong repression of EGFP but had no effect on RFP. Over-expression of a non-PcG protein, DNApol-α50 d shows no effect on PRE-EGFP as both EGFP and RFP expression levels are comparable to those of a. Merge images show comparable RFP signal and cell density. e Total cell lysates from transfected cells a–d were probed with anti-FLAG, anti-GFP and anti-tubulin antibodies on a Western blot. Over-expression of both PC and E(z) shows a drastic reduction in EGFP expression while DNApol-α50 did not significantly change EGFP levels. f Over-expression of PC represses PRE-F.Luc in a dose-dependent manner. Increased repression of F.Luc was observed with increasing amounts (1, 2, 4 ng) of transiently transfected Pc over-expression construct, pActin-Pc. g Knockdown of known members of trxG (trx, ash1, brm, mor) leads to decrease in F.Luc expression in cells transiently transfected with PRE-F.Luc along with pActin-R.Luc (Renilla Luciferase) used as a normalization control. Knockdown of F.Luc revealed strong repression of reporter. dsRNA against LacZ was used as a negative control. Relative F.Luc values, normalized against R.Luc, used as an internal control, recorded at 72 and 96 h after transfection are shown. All knockdowns of trxG genes resulted in significant downregulation of relative F.Luc levels (p < 0.0001) at both time points. Experiments shown in f and g were performed in triplicates in two different sets and were analyzed by student t test f or one-way ANOVA g (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ****p ≤ 0.0001). Error bars represent SEM