Figure 2

Functional implications of HNRNP G and HTRA2-BETA1 in ECin vitro.(A) HNRNP G and HTRA2-BETA1 protein expression in Ishikawa cells transiently transfected with expression and knock-down plasmids; (−) shRNA and (+) expression plasmid for HNRNPG and HTRA2-BETA1; (C) control: empty pCMV-plasmid. HKG:Beta-Actin. Western blot. (B) Influence of HTRA2-BETA1 and HNRNPG mRNA-levels on endogenous ERa-exon7 mRNA splicing. (C) cells transfected with: control: empty pCMV-plasmid; (HTRA2-BETA1+) HTRA2-BETA1-expression-plasmid; (HTRA2-BETA1 −) HTRA2-BETA1-shRNA; (HNRNPG +) HNRNPG-expression-plasmid; (HNRNPG −) HNRNPG-shRNA. RT-PCR. (C)HNRNPG, HTRA2-BETA1, ERa-standard and ERa-exon6 mRNA expression in differently treated Ishikawa cells. (C) control:pCMV-plasmid; (HTRA2-BETA1 +) HTRA2-BETA1-expression-plasmid; (HTRA2-BETA1−) HTRA2-BETA1-shRNA; (HNRNPG +) HNRNPG-expression-plasmid; (HNRNPG −) HNRNPG-shRNA. HKG:RPS18. RT-PCR. (D) Exogenous level of ERa-exon7 splicing pattern. Influence of overexpression (+) and knock-down (−) HNRNPG and HTRA2-BETA1 on alternative ERa-exon7 minigene expression. In untransfected control cells, the reporter gene was alternatively spliced into 4 isoforms, two precisely spliced isoforms are exon7-skipping (137bp) and exon7-inclusion (321bp). Two lariat containing isoforms are: one containing a part of intron sequence between INS-exon2 and -3 (210bp), another containing an additional pseudo-exon from exon7 5’ intron sequence (544bp, all four isoforms were verified by sequencing). RT-PCR. (E,F)ERa exon7 alternative splicing regulation by HTRA2-BETA1 and HNRNPG in Ishikawa cells. (E) ERa-exon7 skipping/inclusion ratio; (F) HNRNPG/HTRA2-BETA1 ratio in differentially treated Ishikawa cells. (C) control:pCMV-plasmid; (HTRA2-BETA1 +) HTRA2-BETA1-expression-plasmid; (HTRA2-BETA1 −) HTRA2-BETA1 shRNA; (HNRNPG +) HNRNPG-expression-plasmid; (HNRNPG −) HNRNPG-shRNA. (G)ERa exon7 skipping/inclusion mRNA ratio difference between HTRA2-BETA1overexpression and HNRNPG overexpression group. (HTRA2-BETA1 +) HTRA2-BETA1 overexpression; (HNRNPG +) HNRNPG overexpression; **ERa exon7 skipping/inclusion ratio between the two groups was statistically significant p= 0.004. PCR-based tests originate on arithmetic mean of triplicate analyses. Student-T-test was applied for data shown in E-G, while statistical significance was assumed at p<0.05 at the two-sided test. Representative gel images in B-D demonstrate one out of three repeats.