Research Paper Volume 15, Issue 24 pp 14591—14606

Figure 4. SRSF7 depletion modulates MDM2-C expression via alternative splicing. (A, B) RNA seq analysis. HDFs (DT2) were transfected with siRNA against either NC or SRSF3 or SRSF7 for 3 days. (A) Principal component analysis. (B) A heatmap of MDM2 transcripts, along with the schematic of each transcript. The values are scaled into z-score. Abbreviations: int: intron; NLS: nuclear localization signal; NES: nuclear export signal. (C) A diagram of exon composition of MDM2-FL with primer positions and exon numbers according to the updated MDM2 gene information (NM_002392.6), along with diagrams of predicted PCR products. (D, E) HDFs (DT2) were transfected with siRNA against either negative control (NC) or SRSF7 for 4 days. (D) RT-PCR and western blot analysis. (E) Diagrams of MDM2 splice variants identified by DNA sequencing, along with a table showing density per base pair of each variant. (^# a point of frameshift occurrence; ^* the position of stop codon). (F, G) HDFs (DT2) were transfected with siRNA against either negative control (NC) or each splicing factor for 3 days. (F) RT-PCR analysis of MDM2-C generation, along with mRNA level of each splicing factor using qPCR. (^**p < 0.01 vs. NC by student t-test). (G) mRNA level of SRSF7 using qPCR. (^**p < 0.01 vs. NC by student t-test). (H–J) HDFs (DT2) were infected by the lentiviruses expressing MDM2-C for 3 days. (H) Western blot analysis. (I) The quantification of cell growth activity (^**p < 0.01 vs. GFP by student t-test). (J) The quantification of SA-β-gal (+) cells along with pictures of stained cells. (^**p < 0.01 vs. GFP by student t-test).