SRP9 and SRP14 knockdowns reduce 7SL and BC200 RNA steady-state levels. (A) MCF-7 cells were transfected with negative control, SRP9, or SRP14 siRNA and harvested following the labeled time points. Whole-cell lysate RNA extraction was performed, and 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 represented as ±SD. (B) Same as (A) but with primers specific to BC200. (C) Same as (A) but with primers specific to tRNASer(GCT). (D) MCF-7 cells for each described transfection condition were harvested and isolated for protein. Whole-cell lysate protein was analyzed by SDS–PAGE and western blot with antibodies specific to SRP14 and SRP9 for monitoring knockdown efficiency, and GAPDH as a loading control. Molecular weight (MW) markers from the protein ladder are presented beside the blot. Representative western blot of three biological replicates was presented. (E) Whole-cell lysate RNA extracted from 48-h negative control and SRP14 siRNA transfected MCF-7 cells were analyzed by RT-qPCR with primers specific to various classes of RNA polymerase III transcripts (I—5S rRNA; II—7SL RNA, BC200 RNA, tRNASer(GCT); and III—7SK RNA). Data were presented as the mean relative RNA level of the SRP14 siRNA samples compared to the negative control across three biological replicates measured in duplicate. Error was shown as ±SD.
