Fig 1: HuR suppression inhibits increase in dCK expression in response to GEMWestern blot analysis of HuR and dCK protein expression in OVCAR5, OVCAR5-shCtrl, and OVCAR5-shHuR cells treated with or without 0.02 µM gemcitabine (GEM) for indicated times.
Fig 2: DDB1 increases the resistance of pancreatic xenograft tumors to GEM. (A) Stably transfected DDB1 shRNA or scrambled-shRNA PDAC cells were injected into nude mice, which were later divided into scramble, shDDB1, scramble+GEM and shDDB1+GEM groups and treated as described in the Methods. (B) The tumor sizes were measured by electronic Vernier calipers. Tumor growth curves were created based on the tumor volume. (C) The heatmap showed differentially expressed genes in shDDB1-transfected (D1) cells and scrambled-transfected (Sc) cells. (D) The expression of DDB1, dCK and Ki-67 was detected in tumor tissue slices from the xenografts using IHC (scale bar, 40 µm). (E) mRNA expression level of dCK in DDB1-silenced or scrambled PDAC cell lines. (F) Effect of DDB1 abrogation on the expression of dCK according to a Western blotting assay. Detailed information of Western blotting figures can be found at Supplement material Figure S1 and Table S3. (G) The correlation between DDB1 and dCK was determined by IHC scores. (H) The possible mechanism of DDB1-mediated GEM resistance and tumor progression due to dCK downregulation and changes in EMT-related genes in patients with PDAC. * p < 0.05, *** p < 0.001.
Fig 3: RNP-IP assays showing increased binding of dCK mRNA to HuR in response to gemcitabineA. HuR protein-bound dCK (left) and SUMO-1 (right) mRNA amounts in A2780 cells grown in the presence or absence of 1µM GEM for 12 h as measured by qPCR (mean +/- SD). B. Left: Western blots of protein lysates prepared from A2780 cells grown in the presence or absence of 1 µM GEM, before immunoprecipitation, and immunoprecipitates prepared with anti-HuR. Three separate IPs were assayed. Right: Western blots of protein lysates before immunoprecipitation, and immunoprecipitates prepared with IgG.
Fig 4: ROM modulates CDA expression and ARAC-induced ?-H2AX. (a) Protein extracted from PER-703A cells incubated with 1 nM ROM for 0, 2, 4, 6 or 8 h, were separated by SDS-PAGE and immunoblotted with antibodies against CDA, DCK and Actin. (b) Histogram of densitometric ratios for CDA:Actin immunoblots (n=2 independent experiments). (c) PER-785A and (d) PER-826A cells were treated with ROM and/or ARAC for 24 h, stained with PI and ?-H2AX and analyzed by flow cytometry. Total cells were defined excluding debris and dead cells; G0-G1, S- and G2-M phases of the cell cycle were gated on PI intensity; ?-H2AX-positive gates were defined by comparison to no drug controls. Graphs display the means of four independent experiments, error bars show s.d. and P-values calculated by one-way ANOVA and Tukey's multiple comparison tests (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Fig 5: HuR nuclear to cytoplasmic translocation following treatment of OVCAR5 cells with gemcitabine (GEM) associated with an increase in cytoplasmic dCK mRNA and proteinA. Western blot analysis for HuR and dCK in cytoplasmic and nuclear protein lysates. GAPDH provided loading control and allowed for quantitative comparison of HuR and dCK at different time points (values indicated beneath HuR and dCK panels). Lamin A/C provides marker for cytoplasmic extract purity. B. qRT-PCR analysis of cytoplasmic dCK mRNA isolated from OVCAR5 cells treated with GEM for different times (mean +/- SD). C. OVCAR5 cells grown in medium +/- GEM and immunostained for HuR. White arrows point to cytoplasmic HuR. D. Sections of human ovarian tumor, collected prior to drug treatment, were immunostained for HuR and dCK. Boxed area is enlarged in lower left corner of each panel in top row.
Supplier Page from Abcam for Anti-DCK antibody