Knock-down of hnRNP K strongly suppresses TGF-β-induced RCC cell invasion.

<p>A. Invasion assay in si-hnRNP K-treated A498 cells. Three individual experiments were performed, and mean ± s.d. is presented; *<i>P</i>, **<i>P</i>, ***<i>P</i><0.05 compared with control (Student’s <i>t</i>-test, n = 3). N.S.; no significant difference; B. Effect of hnRNP K knock-down on TGF-β-induced subcellular localization of hnRNP K expression in A498 cells.</p

Inhibition of proliferation in RCC cells by hnRNP K knocking down.

<p>Expression of heterogeneous nuclear ribonucleoprotein (hnRNP) K in both normal renal proximal tubule cell line (RPTEC) and renal cell carcinoma (RCC) cell lines, and growth inhibition of four RCC cell lines after knock-down of endogenous hnRNP K. As described in the Materials and Methods section, the number of viable RCC cells after treatment with control siRNA or siRNA against hnRNP K (si-hnRNP K) for 0, 24, 48, 72, 96 or 120 h was determined by water-soluble tetrazolium salt (WST-1) assay. A. Total cell lysates were prepared from each cell line and analyzed by western blotting using anti-hnRNP K antibody to detect hnRNP K protein (upper panel). To elucidate equal loaded amounts of total cell lysates, western blotting using anti-β-actin antibody was also performed (lower panel); B. Each RCC cell line transfected with negative control siRNA or si-hnRNP K was harvested 72 h after transfection and total cell lysates were analyzed by western blotting using anti-hnRNP K antibody to evaluate the efficacy of si-hnRNP K; C. Values measured at 0 h with control siRNA or si-hnRNP K were determined as 1. Three individual experiments were performed. Results were presented as mean ± S.D. *<i>P</i><0.05 compared with control (Student’s <i>t</i>-test, n = 3).</p

Functional analysis of exogenous hnRNP K mutants in RCC cells with down-regulated endogenous hnRNP K.

<p>A. Mapping of structural domains of hnRNP K protein. The locations of the nuclear localization signal (NLS), K homologue (KH), K-protein-interactive (KI) and nuclear shuttling (KNS) domains are indicated (upper panel). A putative consensus sequence of the bipartite NLS is defined as (K/R)(K/R)X10-12(K/R)3/5, where X indicates any amino acid and (K/R)3/5 represents at least three of either lysine or arginine out of five sequential amino acids. <i>Arrowheads</i> indicate the conserved amino acid residues in NLS (lower panel); B. Exogenous expression of hnRNP K wild type (wt) or mutant (mt; K21A and R22A) is dominant in the cytoplasm. Single asterisk (*<b>)</b> and double asterisks (**) indicate FLAG-hnRNP K and endogenous hnRNP K, respectively in the left and upper panel. Signals against FLAG-tag are indicated as exogenous expression of hnRNP K; C. Expression of hnRNP K constructs was measured by western blotting with anti-hnRNP K antibody in A498 cells treated with control siRNA or si-hnRNP K. As indicated in the column, these cells were co-transfected with control empty vector, si-hnRNP K resistant wild-type hnRNP K (hnRNP K-wt-siR) or si-hnRNP K resistant mutant hnRNP K (hnRNP K-mt-siR). Anti-calpain antibody or anti-histone H1 antibody was used as loading control for the cytoplasmic or nuclear fraction, respectively; D. Exogenous expression of hnRNP K-wt-siR or hnRNP K-mt-siR significantly suppressed the inhibition of cell invasion by endogenous hnRNP K knock-down in A498 cells. Three individual experiments were performed, and mean ± s.d. is presented; *<i>P</i>, **<i>P</i>, ***<i>P</i><0.05 compared with the second lane treated with si-hnRNP K (Student’s <i>t</i>-test, n = 3); E. Exogenous expression of hnRNP K-wt-siR or hnRNP K-mt-siR reversed the down-regulation of MMP-2 expression induced by endogenous hnRNP K knock-down. Anti-β-actin antibody was used as an indicator of the loaded volume of total cell lysates.</p

Chagnes in subcelluar distribution of hnRNP K under the condition with TGF-β treatment.

<p>A. Evaluation of hnRNP K protein expression in subcellular fractions (C; cytoplasm and N; nucleus) of RPTEC and four RCC cell lines. Expression levels of hnRNP K in cytoplasmic and nuclear fraction are corrected by expression of calpain or histone H1, respectively; B. Estimation of the effect of TGF-β on the subcellular distribution of hnRNP K expression in A498 and Caki-1 cell lines. Each fractioned expression level of hnRNP K is corrected by expression of calpain (cytoplasm) or histone H1 (nucleus).</p

Induction of apoptosis by hnRNP K knocking down in RCC cells.

<p>Measurement of apoptotic cells in A498 (upper panel) and Caki-1 (lower panel) cell lines transfected with control siRNA or si-hnRNP K using flow cytometry. Three individual experiments were performed, and the percentage of apoptotic cells in these cell lines is presented as means ± s.d. *<i>P</i><0.05 compared with control (Student’s <i>t</i>-test, n = 3).</p

Correlation hnRNP K expression alterations with RCC aggressiveness in IHC analysis.

<p>A. Immunoreactivity of hnRNP K in clear cell type (cc) RCC Fuhrman grade 1, 2, 3, and 4; B. A part of the Fuhrman grade 4 micrograph is highly magnified in the right panel; C. Staining scores for hnRNP K in ccRCC tissues with diverse Fuhrman grades; D. Staining scores in primary ccRCC specimens with and without distant metastasis; E. Correlation between cytoplasmic staining and distant metastasis in these ccRCC specimens.</p