Fig. 4
NM1 is required for the activation of Pol II transcription. a Steady state expression levels for NM1 and actin were analyzed on immunoblots of lysates prepared from control (scrRNAi) and NM1-silenced MEFs with an antibody to NM1 and with an antibody against actin. Right panel, densitometric analysis of the immunoblots for semi-quantitative measurements of NM1 steady state protein expression relative to actin. b Immunoblots on lysates from control (scrRNAi) and NM1-silenced MEFs with an antibody to NM1, an anti-pan-myosin 1C antibody against an epitope present in the tail of all three myosin 1C isoforms and with an antibody against actin show marginal off-target effects in the NM1 gene silencing experiment by RNAi. c RT-qPCR analysis of mRNA transcripts on polyA mRNA prepared from MEFs subjected to NM1 gene knockdown (NM1 RNAi) or from MEFs subjected to control siRNA oligonucleotides (scrRNAi). The mRNA levels are relative to β-actin mRNA. Error bars represent the standard deviation of three independent experiments (n = 3). d-g NM1 is required for promoter association of the active Pol II and actin. ChIP and qPCR analysis on chromatin isolated from NM1 knockdown cells (NM1 RNAi) and control cells (scrRNAi), using antibody against NM1, active (4H8) and inactive (8WG16) Pol II, actin, Rpb6 and Rpb8. In all cases, qPCR analysis was performed with primers amplifying (d, f) the mouse Rad9a gene promoter and (e, g) the mouse Rpl19 gene promoter. The values from the qPCR analyses are presented as the percentage of the input signal for each primer pair. All ChIP experiments were successfully repeated three times (n = 3). Error bars represent standard deviations. In panel D, pPol II(4H8) = 0.02 (*); in panel E, pPol II(4H8) = 0.015 (*); in panel F, pactin = 0.05 (*), pRpb6 = 0.025 (*); in panel G, pactin = 0.000522 (***), pRpb6 = 0.0010 (***), pRpb8 = 0.01 (**). Significances were obtained by Student’s t-test, two-sample equal variance. ns = non-significant. In the cartoons above the bars diagrams, arrows point to the approximate position of primers amplifying 150 bp within the promoters bp base pair, ChIP-Seq chromatin immunoprecipitation and deep sequencing, MEFs mouse embryonic fibroblasts, NM1 nuclear myosin 1c, qPCR quantitative polymerase chain reaction