Fig. 3: Validation studies effects of SNPs and splicing factor deregulation on HMMR splicing.

A Schematic representation of HMMR minigene splicing cassette. HMMR exons 3–5 are represented in green and red boxes; dark green lines represent the shortened introns; expected HMMR splice variant transcripts are shown on the figure. B Gel electrophoresis result of the functional splicing assay of the wild-type and mutated HMMR minigene in 293T cells. C Capillary electrophoresis result of the functional assay of the wild-type and mutated HMMR minigene in NCI-H929 cells. The full-length and splice variant transcript are shown as green peaks. The Genescan Liz-1000 size standard is shown as orange peaks. Fragment sizes and relative fluorescent units (RFU; RFU = lg(RFU)) are indicated on the x- and y axes, respectively. D, E HMMR splice variant transcript (D) and protein (E) expression in the NCI-H929 cells stably transfected with PTBP1- and PTBP2-GFP constructs. D shows single-cell RT-PCR capillary electrophoresis results; HMMR splice variant distributions in MM and HD BMSC samples are presented as heatmaps. The color scale for expression values is shown. E shows western blotting and DIC and fluorescence images of NCI-H929 cells overexpressing PTBP1-GFP and PTBP2-GFP. Protein lysates of transfected and parental cells were separated on SDS-PAGE, blotted onto nitrocellulose, and probed with anti-HMMR antibodies. Arrows identifying HMMR-V1/2, V3, and V4 bands are shown.