SRP9/SRP14 knockdown reduces transcriptional activity at 7SL and BC200 genetic loci. (A) qPCR analysis of ChIP enrichment of RNA polymerase III, RNA polymerase II, and a negative control at the 7SL gene locus in MCF-7 cells after 48-h negative control or SRP14 siRNA (SRP9/SRP14 knockdown) transfections. Data were representative of three independent biological replicates, presented as the mean of three technical replicates and error shown as ±SD. Percent of input was calculated from the measurements of 25 ng input DNA. P-values were calculated using an unpaired two-tailed t-test with statistical significance indicated by (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. (B) Same as for (A) but with primers specific to BC200. (C) Same as for (A) but with primers specific to tRNASer(GCT). (D) Relative RNA polymerase III occupancy (solid line) compared RNA polymerase III ChIP enrichments after SRP9/SRP14 knockdown relative to the negative control at each respective time point. Relative RNA level (dashed line) was assessed from whole-cell lysate RNA extracted from MCF-7 cells transfected with negative control, or SRP14 siRNA following the labeled time points. Expression was assessed by RT-qPCR with 25 ng of RNA per reaction. Primers were specific to 7SL, and measurements were presented as the mean of three biological replicates measured in duplicate with error presented as ±SD. (E) Same as for (D) but with primers specific to BC200. (F) Same as for (D) but with primers specific to tRNASer(GCT).
