Increased hnRNP K protein level, not mRNA, in human pancreatic cancer cell lines. Primary fibroblast and pancreatic cancer cell lines were grown at the log phase and harvested. (a) Western blot analysis was performed using hnRNP K and α-tubulin antibodies. (b) Total RNA was isolated and real time RT-PCR analysis was performed. hnRNP K mRNA was normalized against GAPDH mRNA.

siRNA knock down of hnRNP K inhibited tumor cell growth and colony formation. (a) MiaPaCa-2 cells were transfected with negative control (NC) or 50nM or 100nM of hnRNP K siRNAs. Forty eight hours after transfection, cells were harvested and lysed followed by Western blot analysis using hnRNP K and α-tubulin antibodies. (b) MiaPaCa-2 cells were transfected with 50 nM of negative control or 2 different hnRNP K siRNAs. Twenty four hours after transfection, 2×10⁴ cells were seeded into 100 mm plates in triplicate and cell growth were monitored by counting total cell numbers every two or three days; or (c) 1000 cells were seeded into 100 mm plates in triplicate and incubated for 2 weeks to allow colony formation. Then media were removed and colonies were stained with methylene blue solution. The plates were rinsed with water and the colony number was counted.

Changes of the subcellular distribution and the interaction of the mutant p53 and hnRNP K in response to serum and MEK inhibitor. BxPC3 (B) and MiaPaCa-2 (M) cells were either (a) cultured in serum-containing medium or in serum-free medium for 24h, or (b) cultured in serum-free medium for 24h and then treated with U0126 (10 μM) for 1h before harvested and fractionated into cytoplasm (C) and nucleus (N) fractions. Lysates were used in Western blot analyses using hnRNP K, p53, p-ERK, total ERK1/2 or PARP antibodies. (c) Whole cell lysates or the cytoplasmic and the nuclear fractions of HPDE (H), BxPC3 and MiaPaCa-2 cells were immunoprecipitated with hnRNP K or control IgG antibodies followed by immunoblot with p53 and hnRNP K antibodies. (d) BxPC3 and MiaPaCa-2 cells were either cultured in the regular medium or serum-starved for 24h before harvested. Cytoplasmic and nuclear fractions were immunoprecipitated with p53 antibody followed by immunoblot with hnRNP K and p53 antibodies. (e) MiaPaCa-2 cells were cultured in serum-free medium or serum-free medium supplemented with EGF for 24h before harvest. Cytoplasmic and nuclear fractions were immunoprecipitated (IP) with p53 antibody followed by immunoblot (IB) with hnRNP K and p53 antibodies. Subcellular fractions were also used in regular Western blot without immunoprecipitation using the same antibodies. (f) MiaPaCa-2 cells were transfected with negative control or 50nM of hnRNP K siRNAs. Twenty-four hours after transfection, cells were either serum starved for 24h only or treated with U0126 for 1h or kept in serum-containing medium for 24h before harvested and fractionated into cytoplasm and nucleus fractions. Lysates were used in Western blot analyses using hnRNP K, p53, p-ERK, or total ERK1/2 antibodies. (g) MiaPaCa-2 cells were serum-starved for 24h and then treated with U0126 or vehicle control for 1h before harvest. Cytoplasmic and nuclear fractions were immunoprecipitated with either N-terminal epitope or C-terminal epitope p53 antibodies (N-p53 or C-p53) followed by immunoblot with hnRNP K and p53 antibodies.

(a) hnRNP K protein is increased in human pancreatic cancer tissues. H&E staining and immunohistochemistry was performed on a pancreatic cancer tissue microarray using hnRNP K antibody. Representative H&E and immunohistochemistry analysis of the hnRNP K protein in normal, well, moderately- and poorly-differentiated pancreatic cancer tissues were shown. Magnification: 400×. Mean scores of hnRNP K protein expression were analyzed as described in Materials and Methods. The difference between groups was compared by the Student’s t test. (b) Increased cytoplasmic hnRNP K protein in pancreatic cancer cells. Immortalized human pancreatic ductal epithelial (HPDE) cells and pancreatic cancer cell lines BxPC3 and MiaPaCa-2 cells were grown at the log phase and harvested. Cytoplasmic (C) and nuclear (N) fractions were obtained. Western blot analysis was performed using hnRNP K, eIF4E, p53 and α-tubulin antibodies. (c) Transactivation by the mutant p53 in MiaPaCa-2 and BxPC3 cells. BxPC3 and MiaPaCa-2 cells were treated with 10 nM etoposide or vehicle control for 48h before harvest. Western blot analysis was performed using p53, p-p53, p21 and α-tubulin antibodies.