Fig 1: Effect of JNK KD on cell proliferation and colony formation.A, B Effect of JNK down-regulation on basal cell proliferation. Indicated cells (10,000 cells/well for MIA PaCa-2, 5000 cells/well for PANC-1) were cultured for 48 h in 96-well plates in complete medium and analyzed by the MTT assay. Results are shown as optical density and are means (±SD) from three separate experiments with quadruplicate determinations. C–F Anchorage-independent growth. MIA PaCa-2 was not influenced by JNK downregulation (C). JNK2 KD in PANC-1 increased colony formation and size while JNK1 KD showed opposing effects (D, E). Representative images of PANC-1 colony size (left, 2.5× magnification) and colony formation (right, scanned image after MTT staining) are shown in (F). Scale bar: 1 mm. Data are shown as means of three independent experiments of triplicate determinations ±SD. JNK WT cells (WT and Neo clones), JNK1 KD clones, and JNK2 KD clones were analyzed together. ***p < 0.001 compared to JNK WT cells.
Fig 2: Pharmacological JNK inhibition and the effect on pancreatic cancer cell growth.A, B Effect of the JNK inhibitor SP600125 on basal cell proliferation in WT cell lines (A) and after JNK KD (B). Indicated cells (10,000 cells/well) were cultured for 24 h in 96-well plates in complete medium followed by another 48 h in the absence or presence of increasing concentrations of SP600125 followed by the MTT assay. Results are shown as means (±SD) from three separate experiments with quadruplicate determinations compared to untreated control (DMSO only). B Cell proliferation was significantly inhibited at 10 µM SP600125 in wild-type (?) and Neo-17 (o). (C) Reduced JNK activity by SP600125 displayed by phosphorylated c-Jun. SP600125 markedly reduced JNK activity in MIA PaCa-2 and BxPC-3 cells while it was without effect in PANC-1. A representative blot is shown of three independent experiments. *p < 0.05 compared to JNK1 and JNK2 KD cells.
Fig 3: Orthotopic in vivo xenograft.A Tumor volume. JNK2 KD cells showed increased tumor volume, especially for M-2–12. B, C H.E. staining revealed solid pancreatic tumors in all xenografts. In control cells and JNK1 KD cells (M-1–18), the tumor was restricted to intrapancreatic growth while JNK2 KD clones regularly infiltrated surrounding organs. Tumors (TU) of M-2–12 (left) and M-2–24 (right) showed infiltration of the stomach (STO) and duodenum (DUO). The dashed line represents the tumor infiltrative line. This infiltrative pattern was not seen in control cells and JNK1 downregulated cells. Scale bar: 200 µm. *p < 0.05.
Fig 4: Effect of MAPK inhibitors on IL-1ß-induced COX-2 expression. Synovial fibroblasts were pretreated with or without the MEK inhibitor U0126 (20 µM), ERK1/2 inhibitor FR180204 (50 µM), JNK inhibitor SP600125 (10 µM), and p38 inhibitor SKF86002 (10 µM) for 1 h and subsequently stimulated with IL-1ß. After stimulation for 6 h or 48 h, we determined COX-2 expression (A) and prostaglandin E2 release (B). IL-1ß-induced COX-2 expression and prostaglandin E2 release were significantly reduced by the inhibitors. Results have been represented as mean ± SE from biological triplicates. *P < 0.05.
Fig 5: Analysis of selected cytokines after a 3-day s-µg-exposure: Transcriptional and translational IL-6 analysis: Quantitative gene expression levels of IL-6 (A) and intracellular IL-6 levels (B), release of IL-6 (C). Transcriptional and translational IL-8 analysis: Quantitative gene expression levels of CXCL8 (D) and intracellular IL-8 levels (E), release of IL-8 (F). Transcriptional and translational MCP-1 analysis: Quantitative gene expression levels of CCL2 (G), intracellular MCP-1 levels (H), release of MCP-1 (I). Quantitative gene expression levels of TNFA (J). Transcriptional and translational NF-?B p65 analysis: Quantitative gene expression levels of RELA (K) and intracellular NF-?B p65 levels (L). Quantitative gene expression levels of ICAM1 (M). Transcriptional and translational JNK1 analysis: Quantitative gene expression levels of JNK1 (N) and intracellular JNK1 protein levels (O). Full-length blots of cropped Western blot images are presented in Supplementary Fig. S5. *p < 0.05 1g vs. RPM; #p < 0.05 AD vs. MCS.
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