Fig 1: JAK inhibition and 5-ASA counteract the mutagenic effect of IL-6, but only JAK inhibition reduced PMN-induced MSI. (A) Treatment of HCT116 + chr3-[CA]26 with tofacitinib, a JAK inhibitor, neutralized IL-6-induced frameshift mutations. Total cell number decreased upon treatment with IL6 and/or tofacitinib. (B) Similarly, 5-ASA also reduced IL-6 induced frameshift mutations in HCT116 + chr3-[CA]26. 5-ASA decreased cell count, which was further diminished upon IL6 treatment. 5-ASA decreased cell number both of untreated and IL-6-treated cells. (C) 1 × 104 EGPF-negative HCT116 + chr3-[CA]26 were sorted in 24 wells. Twenty-four hours later, cells were treated with 5 mM 5-ASA and/or 2 µM tofacitinib before the addition of PMA-activated PMNs at an effector:target ratio of 75:1. Flow cytometric analysis was performed 7 days later. Tofacitinib counteracted PMN-induced frameshift mutations (which is presented as fold change to cells treated with PMA-activated PMNs only), whereas 5-ASA failed to do so. No additive effects were present.
Fig 2: PMN-released cytokines may induce frameshift mutations, internal ROS production and DNA oxidation. (A) 2 × 106 PMNs were activated with 0.5 nM PMA for 16 h and supernatants were subjected to a bead-based cytokine assay (Biorad) measured on a Bio-plex 200 instrument (Biorad). Treatment with 25 ng/ml IL-6, IL-8 or TNF-α decreased cell count and increased MR in HCT116 + chr3-A10 (B) or HCEC-1CT-[CA]26 (C). (D) Culture of HCEC-1CT with 25 ng/ml IL-6, IL-8 or TNF-α induced internal ROS production (as measured by DCFDA and expressed as relative fluorescent units, RFU) in a time-dependent manner, with the highest upregulation at 3 h. (E) HCT116 + chr3 cells were exposed to IL-6, PMA or H2O2 for 24 h. The formation of oxidized purines (8-oxo-dG) was analyzed with and without FPG. DNA migration was evaluated using single cell gel electrophoresis and computer-aided analysis. Bars indicate means ± SD of results obtained from 150 cells from three slides each experimental condition (t-test).
Fig 3: IL-6 Sandwich Immunoassay Procedure Based on PLNPs
Fig 4: IL-6 sandwich immunoassay in undiluted human serum ([Abc] = 10 μg·mL–1, [Abd] = 2.36 μg·mL–1, n = 6). (a) Persistent luminescence decays. RLU = Relative luminescence units. (b) Normalized integrated area under the decay curves shown in Figure 9a versus IL-6 concentration. (c) Unpaired and two-tailed t-test obtained from the data analysis of Figure 9b.
Fig 5: BZA-dependent inhibition of gp130/STAT3 signalling demonstrates synergistic activity with the standard-of-care chemotherapy.A Flow cytometry analysis of Annexin V-FITC (AV) and TO-PRO-3-iodide stained LIM2405 cells treated with 15 μM BZA, 25 μM 5-fluorouracil (5FU), 50 μM oxaliplatin (OX), 5 μM SN38 alone and in combination (BZA + 5FU, BZA + OX and BZA + SN38) in the presence of 30 ng/ml IL-11 for 24 hours. Error bars are mean ± SEM of n = 3 replicates. Student’s unpaired t-test: ****p < 0.0001, ***p < 0.001, **p < 0.01. B Schematic representation of organoid experiment workflow. Tumour biopsies were taken from the sigmoid and ascending colon of patients with stage II and stage III colon cancer, processed into single cancer cells and then cultured as 3D patient-derived organoids (PDO) to be tested using novel drug combinations. C Western blot of pSTAT3 and total STAT3 protein levels in PDO 53 T stimulated with IL-6 and IL-11 for 15 min and 24 hours. D Western blot of pSTAT3 and total STAT3 protein expression in PDO 27 T stimulated with IL-6 and IL-11 for 15 min and 24 hours. E Western blot of ERα protein in PDO 53 T and PDO 27 T treated with 3 μM BZA for 7 days. F, G Cell viability, Bliss excess scores and HSA excess scores of the indicated concentrations of BZA + 5FU, BZA + OX and BZA + SN38 in PDO 53 T and 27 T over 7 days. High synergy scores are represented in red and low scores indicating antagonistic effects are represented in blue.
Supplier Page from Thermo Fisher Scientific for Recombinant Human IL-6 Protein