Cell viability and morphology are maintained in the absence of SR expression. (A) Representative time course of siRNA-mediated SRA knockdown in HeLa Cas9 WT cells assessed via immunoblot for SRA (target), β-tubulin (cytosolic control), and TRAPα (ER membrane control) after fractionation of cells into the cytosol (C) and ER compartments. Nontargeting siRNA control = ctrl. (B) As in (A), analysis of SRA mRNA levels by RT-qPCR from total RNA extracts. Data represent expression levels relative to 0 h and were normalized to GAPDH. n = 2 biological replicates. Data are means ± SD. (C) Representative immunoblot analysis of subcellular distributions ([C] cytosol, [ER] endoplasmic reticulum) of SR subunits (SRA and SRB), SRP subunit (SRP54), cytosolic protein (β-tubulin), and ER-resident protein (TRAPα) in HeLa Cas9 WT and SRB KO2 cells nontransfected (−) or transfected with nontargeting (ctrl) or SRA siRNA, 96 h post-transfection. (D) RT-qPCR analysis of SRA and SRB mRNA levels for experimental conditions in (C). Nontransfected wild-type expression levels were set as 1 after normalization to GAPDH. n = 3 biological replicates. Data are means ± SD. (*) Indicates P-value ≤0.01. (E) Representative immunofluorescence micrographs depicting β-tubulin (red) and TRAPα (ER marker) (green) for HeLa Cas9 WT and SRB KO2 cells transfected with either nontargeting (ctrl) or SRA siRNA, assayed 96 h post-transfection. DAPI nuclear stain is included in merged images (blue). Scale bar, 20 µm. (F) CFSE analysis of cell doubling rates assessed by flow cytometry over 3 d of cell culturing for cell conditions as in (C). Data are representative of three biological replicates. (G) [35S] methionine/cysteine incorporation over a 7.5 min labeling period for cell conditions as in (C). Data are presented as mean ± SD for three biological replicates.
