Fig 1: CAFR-derived IL8 activated the NF-?B signaling pathway to upregulate UPK1A-AS1.A Top 20 upregulated pathways were plotted based on the enriched gene ratio and p value in Panc-1 cells treated with CAFR1-CM compared to CAFS1-CM. B UPK1A-AS1 expression in Panc-1 and MiaPaCa-2 cells treated with IL8 (100 ng/ml) alone or IL8 and CAPE (2 µM) together for 3 days. C Western blot analysis of I?Ba, p-I?Ba, p65, and p-p65 protein expression in Panc-1 and MiaPaCa-2 cells treated with CAFR1-CM or IL8. A neutralizing antibody against IL8 was used to deplete IL8 in CAFR1-CM. D Western blot analysis of p65, and p-p65 protein expression in PDAC tissues from platinum-resistant patients and platinum-sensitive patients. E Luciferase reporter assays of Panc-1 cells transfected with a reporter plasmid containing the UPK1A-AS1 promoter, and treated with IL8 or p65 depletion. F, G A conserved p65-binding motif was predicted by JASPAR and schematic images of the potential p65 motif binding sites in the promoter of UPK1A-AS1 are shown. H ChIP analysis of the p65 occupancy at the promoter of UPK1A-AS1 in Panc-1 cells. I Luciferase reporter assays of Panc-1 cells treated with IL8 and transfected with reporter plasmids containing P1, P2 and P3 deletions in the UPK1A-AS1 promoter. The results are presented as the mean ± SD of three technical replicates. **P < 0.01; ***P < 0.001; ns no significance.
Fig 2: CAFR facilitated oxaliplatin resistance by activating the expression of the lncRNA UPK1A-AS1 in pancreatic cancer.A Heatmap of the differentially expressed lncRNAs (|Log2FC | >1, Q value < 0.05) in the lncRNA sequencing data of Panc-1 cells treated with CAFS1-CM or CAFR1CM. Three biological replicates of each CAF cell line were used for the RNA sequencing. B Volcano plot showing the differentially expressed lncRNAs between the groups. UPK1A-AS1 expression levels in Panc-1 (C) and MIAPaCa-2 (D) cells treated with CAFS-CM and CAFR-CM of four different chemosensitive and chemoresistant CAF cell lines. E UPK1A-AS1 expression levesl in Panc-1 and MIAPaCa-2 cells cultured under IL8 (100 ng/ml) or CAFR1-CM with an anti-IL8 neutralizing antibody (250 ng/ml) for 3 days. UPK1A-AS1 was overexpressed or knocked down in Panc-1 cells and oxaliplatin was given. F–H CCK-8 assay, (I–J) colony formation assay and (K–L) flow cytometry apoptosis analyses were performed to evaluate the chemoresistance of Panc-1 cells in each group. The results are presented as the mean ± SD of three technical replicates. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig 3: UPK1A-AS1 overexpression correlates with a poor prognosis in PDAC.A Representative image of ISH of UPK1A-AS1 and IHC for IL8. Scale bars = 200 µm. Quantification of IL8 (B) and UPK1A-AS1 (C) staining in tumors from platinum-sensitive and platinum-resistant patients. The results are presented as the mean ± SD of 21 and 54 biological replicates, respectively. D Correlation between UPK1A-AS1 expression and IL8 protein levels in PDAC tissues. E ELISA analysis of the serum IL-8 levels in platinum-resistant patients and platinum-sensitive patients. The results are presented as the mean ± SD. F Kaplan–Meier survival curves of patients who received platinum-based chemotherapy with high or low UPK1A-AS1 expression. G Kaplan–Meier survival curves of patients who received chemotherapy without platinum with high or low UPK1A-AS1 expression. H Graphical illustration of CAFR derived IL8 mediating UPK1A -AS1 activation in PDAC cells to induce oxaliplatin resistance. **P < 0.01; ***P < 0.001.
Fig 4: Paracrine IL8 was essential for CAFR-induced oxaliplatin resistance in tumor cells.A Cytokine antibody array of CAFS1-CM or CAFR1-CM. CAFR1-CM with different neutralizing antibodies was used to culture Panc-1 (B) and MIAPaCa-2 (C) cells for 3 days. After 48 h of oxaliplatin exposure, cell viability was measured by CCK8. D Panc-1 and MIAPaCa-2 cells were cultured with different concentrations of human recombinant IL8 for 3 days and subsequently treated with oxaliplatin for 48 h. Cell viability was measured by CCK8. E ELISA analysis of the IL-8 levels in CAFS-CM and CAFR-CM of four different chemosensitive and chemoresistant CAF cell lines. Panc-1 and MIAPaCa-2 cells were cultured with IL8 (100 ng/ml) or CAFR1-CM with an anti-IL8 neutralizing antibody (250 ng/ml) for 3 days. Colony formation assay (F, G) and flow cytometry apoptosis analysis (H, I) were performed to evaluate the chemoresistance of pancreatic cancer cells in each group. The results are presented as the mean ± SD of three technical replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ns no significance.
Fig 5: The CAFR/IL8/UPK1A-AS1 axis facilitated NHEJ pathway via the scaffold function of UPK1A-AS1.IL-8 (100 ng/ml), an anti-IL8 neutralizing antibody (250 ng/ml), and CAFR-CM were given to each group as indicated for 3 days. Panc-1 and MIAPaCa-2 cells were treated with 50 µM oxaliplatin and 30 µM oxaliplatin, respectively, in all experiments. A Oxaliplatin-induced DNA damage in control and UPK1A-AS1 knockdown Panc-1 and MIAPaCa-2 cells was measured by neutral comet assay. Scale bar = 10 µm. B Levels of oxaliplatin-induced DNA damage, quantified by the tail moment in the neutral comet assay. In total, 70 cells per group are counted. C Representative pictures of ?H2AX-positive foci in each group. Scale bar = 10 µm. D Quantification of the number of ?h2ax positive foci in each group. At least 40 cells per group are counted. E NHEJ-mediated DNA repair efficiency was measured by a pimEJ5-GFP reporter assay, in Panc-1 and MIAPaCa-2 cells transfected with control vector or UPK1A-AS1, and three technical replicates were performed. F Levels of Ku70 and Ku80 in the complex pulled down by Ku80 (upper panel) and Ku70 (lower panel) specific antibodies, in Panc-1 cells transfected with control vector, UPK1A-AS1 and mutated UPK1A-AS1 of region A and region B. G Fluorescence assessment of UPK1A-AS1 and ?H2AX colocalization in Panc-1 cells. Scale bar = 5 µm. Representative images of western blotting analyses of DNA-PKcs, Ku70, Ku80 and XRCC4 in CNETs (H) and total proteins (I) of Panc-1 cells. The results are presented as the mean ± SD. **P < 0.01; ***P < 0.001.
Supplier Page from Abcam for Human IL-8 ELISA Set (without plates)