Fig. 7: IL-34 increases IL-6 RNA and protein expression in both TICs and LPMCs.

TICs (A) and LPMCs (B) were either left unstimulated (Unst) or stimulated with increasing doses of human IL-34 (25–100 ng/ml) for 6 h (left panel) or 48 h (right panel). Left panel: IL-6 RNA transcripts were analyzed by real-time PCR, levels were normalized to β-actin, and data are expressed as mean ± SEM of eight experiments. Right panel: IL-6 secretion was measured in the cell-free supernatants of TICs and LPMCs treated as above, and the data are expressed as mean ± SEM of eight experiments. C Intestinal TICs were isolated from tumoral samples of patients with CRC and transfected with a specific IL-34 antisense oligonucleotide (IL-34 AS) or with a scrambled antisense oligonucleotide (Src AS) (both used at 2 μg/ml) for 24 h. IL-34 and β-actin were analyzed by western blotting. One of three independent experiments is shown. D IL-6 secretion was measured in the supernatants of TICs isolated from tumoral samples of three patients with CRC and treated as described in C for 48 h. Horizontal bars indicate median values. E Intestinal TICs were isolated and treated as indicated in C and analyzed for the percentage of live CD45 + CD68 + HLA-DRII + CD163 + CD206 + cells expressing IL-6 (each point in the graph) by flow cytometry. Horizontal bars indicate the median values. Right panel: representative histogram showing IL-6 expression in live CD45 + CD68 + HLA-DRII + CD163 + CD206+ TICs isolated from tumoral samples of one patient with CRC, transfected with Src AS or IL-34 AS and analyzed by flow cytometry. Histogram with the respective isotype control antibody is also shown.