Si ns

Human osteosarcoma U2OS cells and human cervical cancer HeLa cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). U2OS and HeLa cells were grown in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
SRSF3-specific siRNA (si-SRSF3, cat. No. M-030081–00) and SRSF1 (SF2/ASF)-specific siRNA (si-SRSF1, cat. No. M-018672–01) were purchased as a siGenome SMARTpool siRNA from Dharmacon (Lafayette, CO). A non-targeting siRNA (si-NS) obtained from Dharmacon, with 52% GC content (cat. No. D-001206–08–20), was used as an siRNA control. The SRSF3-specific siRNA s12732, targeting a splice junction of SRSF3 exon 2 and exon 3, was purchased from Ambion (Austin, TX, USA). Cell transfection and siRNA knockdown assays were described in our previous publication (28 (link)).

The non-specific control siRNA (siNS; #D-001810-10; Dharmacon, Inc., Chicago, IL, USA) and human ELK3 siRNA (siELK3; #L-010320-00-0005; Dharmacon, Inc.) were obtained from Dharmacon (GE Healthcare Life Sciences, Chalfont, UK). The siRNAs (100 nM) were transfected into LEC using Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and cells were collected at 48 h following transfection for further analysis. Total cellular RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc., Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. Total RNA (2 µg) was used for single-stranded cDNA synthesis with OmniScript^® reverse transcriptase (Qiagen GmbH, Hilden, Germany). RT-qPCR was performed using the CFX96 Touch™ Real-Time PCR Detection system (Bio-Rad Laboratories Inc., Hercules, CA, USA) using SYBR-Green I (Qiagen Inc., Valencia, CA, USA). The final volume of PCR mixture was 20 µl, comprising 1 µg cDNA, 10 pmol forward and reverse primers and 10 µl of 2X SYBR mix. The reactions were performed for 40 cycles using optimized cycling conditions (denaturation at 95°C for 1 min, annealing at 55°C for 30 sec and extension at 72°C for 30 sec). RT-qPCR data was processed based on the 2^−ΔΔCq method (13 (link)). The primers used in this analysis are listed in Table I.

HeLa cells were transfected with Dharmacon si-NS or si-SRSF3 twice with an interval of 48 h. The cells at 48 h after the second transfection were treated with 10 μg/ml of actinomycin D for 0, 1, 2, 4 and 8 h (50 (link),51 (link)). Total RNA was extracted at each time point and treated by DNase TURBO (Life Technologies). The DNase-treated total RNA at 1 μg was reverse-transcribed and quantified by TaqMan real-time PCR for the remaining RNA levels of SRSF3 and GAPDH (Applied Biosystems, Life Technologies). Relative SRSF3 RNA expression levels at individual time points were determined by ΔΔC_T method (52 (link)). After normalizing to GAPDH RNA, the SRSF3 RNA decay rates were calculated by setting the RNA levels at 0 h as 100% for both si-NS and si-SRSF1 groups. Exponential fitting curves were determined by the logarithmic least squares method (50 (link)).