Fig. 6
Ixa + Dina induced apoptosis is mediated by upregulated JNK signaling. A Venn diagram showing overlapping genes with at least 2-fold change upregulation or downregulation after treatment with Ixa + Dina versus DMSO control in PDXO1, 11 and 12. B Log2 fold changes of common differentially expressed genes between Ixa + Dina and DMSO groups for PDXO1, 11, and 12. Dotted lines indicate cut-off at fold change of 2. C Heatmap of directed global significance scores based on NanoString gene set analysis. D and E Immunoblot of JNK pathway proteins (D) and quantitative RT-PCR analysis of c-Jun transcriptional activity (E) after treatment with ixazomib and dinaciclib in HCC PDXO1 and 11. Data presented as mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared to respective DMSO controls. F Immunofluorescence co-staining of c-Jun and cleaved caspase 3 in Ixa + Dina treated HCC PDXO1 in the presence of siRNAs targeting c-Jun (siJUN) and JNK (siMAPK8). Green fluorescence indicates c-Jun expression; Red indicates cleaved caspase 3; Blue indicates DAPI staining. Scale bar = 50 μm. G Quantification of tumor cells that co-express both c-Jun and cleaved caspase 3 (cJun+/CC3+) per field. Data presented as mean ± SD, n = 10. **, P < 0.01; ***, P < 0.001. H Relative viability of Ixa + Dina treated PDXO1 and PDXO11 in the presence of a JNK inhibitor (SP600125) at 10 μM (SP10), 20 μM (SP20), and 30 μM (SP30) concentrations. Data presented as mean ± SD, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All statistical analyses were performed using two-tailed Student’s t test. I Immunoblotting of apoptotic markers after Ixa + Dina and JNK inhibitor treatment in HCC PDXO11. J Correlation (Pearson’s r) between basal JNK pathway expression and Ixa + Dina sensitivity (represented by combination IC50 values of ixazomib) in HCC PDXOs. JNK pathway expression was determined using the sum of p-JNK/JNK and c-Jun protein expression quantified from immunoblot analysis