Fig 2: HMMR splice variant transcripts are frequently and recurrently expressed in different subpopulation of cells from MM patients.This figure displays HMMR splice variant expressions at a single-cell level. MM or HD cells were sorted by index sorting followed by RT-PCR DNA fragment analyses and capillary electrophoresis. A, B display HMMR splice variant expression at the single-cell level in MM-PCs and myeloid cells, while C and D display the expression of those variants in autologous MM BMSCs and HD BMSCs. A, C show HMMR splice variant co-expression levels. In these figures, the x axes display cells and the y axes relative fluorescent units (RFU) of the PCR products. PCR product RFU = lg(RFU). RFU is a unit of measurement calculated relative to the size standards included in each reaction. B, D The overall distribution of the HMMR splice variants in different subpopulations of MM and HD samples are presented as heatmaps. The color scale for expression values is shown on the right.

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.

Fig 4: HMMR was overexpressed in PCa and associated with poor prognosis.A, B Analysis of HMMR mRNA expression in PCa and normal tissues (GSE29079, 38241). C Analysis of HMMR expression in 51 paired normal and PCa samples in TCGA. D, E HMMR mRNA expression in local PCa and metastatic tissues (GSE6919, 8511). F Biochemical recurrence risk assessment in TCGA stratified by HMMR expression. G, H Cox regression analysis (G: univariate; H: multivariate) to assess the correlation of HMMR expression and pathological indicators. I qRT–PCR analysis of HMMR mRNA in 10 paired PCa and adjacent tissues. J, K Representative IHC analysis of HMMR expression in PCa and adjacent tissues (J) and IHC quantification of HMMR expression in PCa and adjacent tissues (K), scale bar:100 μm. L Western blotting analysis of HMMR in 10 paired PCa and adjacent tissues. Data were presented as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: HMMR was up-regulated in LUAD. (A, B) The expression of HMMR in LUAD examine by TCGA databases. (C–I) The correlation between HMMR expression and clinical features in LUAD. (J) The ROC curve of HMMR in LUAD. (K) The prognosis of HMMR in LUAD examine by TCGA database. Overall survival (os), disease specific survival (DSS), progression-free survival (PFS), SD (stable disease), and PD (progressive disease). Ns, P > 0.05; ***P < 0.001.