Fig. 5

Nascent A-ROD enhances DKK1 transcription elongation. a RT-qPCR assaying the steady-state chromatin-associated RNA levels of A-ROD, DKK1 and two unrelated transcripts, using several amplicons, at 16 h post-transfection of 50 nM ASOs targeting A-ROD intronic sequences (see also Supplementary Fig. 5a-b and Supplementary Fig. 7a). Error bars represent standard deviations from three independent experiments (n = 3 biological replicates, *p  < 0.05 and **p < 0.01, two-tailed Student’s t-test). b RT-qPCR assaying the steady-state nucleoplasmic RNA levels using several amplicons at three time-points post-transfection of pooled intronic ASO sequences transfected at 100 nM final concentration; either ASO 3 and 4 targeting the first intron of A-ROD or ASO 5 and 6 targeting the second intron; or the standard negative control ASO. Error bars represent standard deviations from three independent experiments (n = 3 biological replicates, *p < 0.05, two-tailed Student’s t-test). c Normalized RT-qPCR levels in the BrU-labeled nascent chromatin-associated RNA (BrU IP done from the chromatin-associated RNA fraction) in control and A-ROD intronic-ASO treated cells. Error bars represent standard deviations from three independent experiments (n = 3 biological replicates, *p < 0.05, two-tailed Student’s t-test). d Transcriptional effect assayed by extracting the 5′ to 3′ BrU-IP recovery ratio in control and A-ROD intronic-ASO treated cells. e ChIP for P-Ser2 Pol II in control MCF-7 cells, cells treated with siRNA against A-ROD (here double knock-down by cotransfecting pooled A-ROD.5′si and A-ROD.3′si2) and MCF-7 cells treated with the A-ROD intronic-ASO3 (here for 24 h). Error bars represent standard deviations from three independent experiments (n = 3 biological replicates, *p < 0.05 and **p < 0.01, two-tailed Student’s t-test). f DKK1 transcriptional effect in control, A-ROD siRNA and intronic-ASO3 treated cells, assayed by extracting the ratio of 5′ to 3′ P-Ser2 Pol II ChIP signal depicted in (e)