Fig 2: Tumorigenic expression of PIM1 and RBMY in primary HCC tissues. (A) PIM1-specific IHC on paraffin-embedded tissue sections of primary HCC. PIM1-C+–expressing tissue showed PIM1-positive signal only in the cytoplasm. PIM1-Mix pattern comprised cells expressing both nuclear and cytoplasmic PIM1 (arrowheads) and also PIM1-C+ (arrows). (B) PIM1 and RBMY double-stained IFA was accomplished on primary HCC tissues using anti-PIM1 and SRGY clone 1 antibodies. Yellow arrows, PIM1-Mix/RBMY-N+; white arrows, PIM1-Mix/RBMY-Mix; and white arrowheads, PIM1-C+/RBMY-C+. (C) PIM1- and RBMY-specific IHC on serial paraffin-embedded tissue sections of primary HCC. BV, blood vessel; DAPI, 4',6-diamidino-2-phenylindole.

Fig 3: Increasing cytoplasmic accumulation of RBMY by PIM1. (A) Subcellular fractionation coupled with Western blot assay on Huh-7 cells co-expressed with proteins indicated. Detection of Lamin B1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed as for controls of the purities of nuclear (Nuc) and cytoplasmic (Cyt) fractions, respectively. Bar plot represents the densitometry analysis of GFP-RBMY-p. The densities of GFP-RBMY-p in nuclear and cytoplasmic fractions were normalized to the intensities of their corresponding markers, Lamin B1 and GAPDH, respectively, and then divided by the total nuclear + cytoplasmic densities of GFP-RBMY-p to obtain a percentage value. (B) Fluorescence imaging distinguished the impact of the indicated kinases on the distribution of GFP-RBMY in Huh-7 cells. The arrowheads and arrows indicate the RBMY-N+ and RBMY-C+/Mix patterns, respectively. (C) Comparison of the subcellular distribution of GFP-RBMY upon co-expression with HA-PIM1, HA-MEK2, or HA-CK1e kinases in HCC cell lines. The microscopic signals of GFP-RBMY-Mix/C+ and N+ were plotted as a mean percentages of the cell population (±SDs). (D) Fluorescence microscopy analysis showed the N+ (arrowhead) and C+/Mix (arrow) patterns of GFP-RBMY in Huh-7 cells upon treatment of kinases inhibitors. (E) Western blot analysis exposed the expression level of GFP-RBMY in nuclear and cytoplasmic fractions of HCC cells treated with dimethyl sulfoxide (DMSO) solvent control or kinase inhibitors. Cells were incubated with the indicated inhibitors (30 µmol/L per well) after attachment. Transfection of GFP-RBMY was performed 24 hours post-treatment. Cells then were refreshed with medium containing the indicated inhibitors and cultured for another 24 hours before subcellular fractionation. Quantitative densitometry analysis was accomplished as mentioned previously. DAPI, 4',6-diamidino-2-phenylindole; RBMY-p, phosphorylated RBMY.

Fig 4: RBMY plays multiple oncogenic functions in HCC cell lines. Subcutaneous HCC xenograft nude mice models engrafted with (A) siRNA-transfected and (B) shRNA-transduced Huh-7 cells were established. The endogenous RBMY was depleted with (A) siRBMY or (B) shRBMY. (B) Restoration of RBMY was achieved by overexpression of HA-RBMY. The xenograft tumor growth in nude mice was measured and compared with the control groups, (A) siCtrl and (B) shCtrl. The silencing efficacy of RBMY was assessed by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a loading control. (C) Tumor volume determined the xenograft growth and is plotted as means ± SDs. Statistical significance was evaluated with the Student t test. (D) Soft agar colony formation of stable Huh-7 transfectants was reported as means ± SDs (Student t test). (E) Scratch wound healing assay showed the migratory ability of indicated cells. Migration distance was assessed and depicted as means ± SDs (Student t test). (F) Transwell invasion assay showed the invasive capacity of the indicated cells. Bar graph (means ± SDs, Student t test) represents the number of cells migrating across Transwell membranes. RBMY-p, phosphorylated-RBMY.

Fig 5: Regulation of RBMY oncogenicity by its upstream kinases. (A) AlamarBlue cell viability assay evaluated the survival and proliferation ability of the indicated transfectants. Cell viability represented by the measured absorbance at 570 nm was plotted as the means ± SDs after normalization to the corresponding values at 0 hours. *P < .005 vs the GFP+HA control was determined by the Student t test. (B) 3D spheroid invasion assay of indicated transfectants. The invasiveness of spheroids grown in a 3D matrix was determined by the formation and enrichment of spindle-like protrusions (black arrows). Representative micrographs acquired before and after the formation of protrusions from spheroid bodies are shown. The mean percentage of the invaded spheroids was calculated and is shown in a bar graph. The error bars indicate the SDs of 3 wells in 2 independent experiments, and significance was evaluated by the Student t test. (C) A wound healing assay showed the migration efficacy of the indicated transfectants. A representative image of each group before (0 h) and after (48 h) cell migration is displayed. Cells expressing GFP and HA served as controls. The migration distances were measured and are reported as the means ± SDs (Student t test). The data shown are representative of 3 independent experiments. (D) Transwell invasion assay to determine the invasive potential of the indicated transfectants. The numbers of invaded cells are presented as the means ± SDs of 4 independent experiments, and significance was evaluated by the Student t test. (E) Scratch wound healing assay showed the migratory abilities of SNU-387 cells cotransfected with plasmids indicated. The migration distances were depicted as the means ± SDs (Student t test). (F) Transwell invasion assay on the indicated transfectants. The number of invaded cells was plotted as means ± SDs and evaluated by the Student t test for statistical significance. (G) Scratch wound healing and Transwell invasion assays on cells transfected twice with siCtrl/siRBMY, then once with HA/HA-PIM1 expression plasmids. Migration distance (means ± SDs) and number of invaded cells (means ± SDs) were analyzed 48 hours postseeding, and differences between groups were estimated by the Student t test.