Figure 2.

Silencing of TFL1 or TFL2 was lethal and reduced SL RNA abundance. (A) Cumulative culture growth curves were obtained for TFL1 and TFL2 silencing in the absence and presence of doxycycline (dox), the gene knockdown-inducing small molecule. For each knockdown, two independently derived cell lines were investigated. (B) Analysis of total RNA from one cell line for each knockdown in uninduced state (0) and after 3 days of doxycycline induction. TFL1 and TFL2 and, as a control, α tubulin (ATUB) mRNAs were analyzed by reverse transcription (RT) of oligo-dT and semi-quantitative PCR. To confirm equal RNA content, rRNA was visualized by ethidium bromide staining after agarose gel electrophoresis. Numbers on the right were derived from RT-qPCR assays and specify the percentage of the targeted mRNA that remained relative to ATUB RNA in gene-silenced cells. (C) Relative abundances of SL RNA and U2 snRNA in total RNA of uninduced and 1, 2, and 3 days TFL-silenced cells were determined by primer extension assays, using a SL RNA- and a U2 snRNA-specific primer in the same reactions. Numbers on the bottom were determined by densitometry and specify the strength of the SL RNA signal normalized by the U2 snRNA signal for each lane. The value in uninduced cells was arbitrarily set to 100. The numbers represent the average of two independent experiments. (D) Lysates of uninduced cells and of cells in which either TFL1 or TFL2 was silenced (KD) for 3 days were immunoblotted, detecting phosphorylated (pRPB1) and unphosphorylated RPB1.