Fig. 1: MPM cells secretion chemoattract and activate lung fibroblasts.

A Cytokine,- chemokine and acute phase proteins profiling was performed on three patient-derived MPM cell lines and log2 fold changes in expression over mesothelial cells (Met-5A) represented as a heatmap. B Consistent log 2-fold changes in 5 cytokines from (A) were represented as bar graphs. Data shown are from a representative repeat. C MRC-5 fibroblasts were placed in the upper part of a Boyden chamber with conditioned media from the indicated MPM cell lines placed in the bottom chambers. Data shown are the mean ± SD of 3 biological replicates normalised to results obtained with CM from Met-5A cells. Two-way ANOVA with Dunnett’s multiple comparison tests were performed to assess statistical significance (****P < 0.001). D MRC-5 fibroblasts incubated with CM from individual MPM cell lines (ie AF 33 are NFs treated with CM from Meso 33) or Met-5A cells (NFMet) for 72 h were stained with Alexa488-Phalloidin (green) and Hoechst (blue) prior to confocal microscopy. Left panel: exemplar image. Scale bar: 100μm. Right panel: Cell area in pixels was calculated for N = 30 cells using Image J and plotted as a Boxplot. Red line represent median. Statistics: Unpaired t-test (****P < 0.001). E MRC-5 cells were seeded in CMs from the indicated MPM cell lines (Meso) or Met-5A cells for the indicated time. Cell lysates were analysed by Western Blotting for the indicated proteins. Upper panel: Data shown are representative of 4 biological replicates. Lower panel: Optical densitometry quantification of 4 biological replicates. F MRC-5 cells seeded for 72 h in CMs from indicated cell lines were subjected to a Caspase 3/7 substrate-based activity assay in 3D cultures. Data are mean ± SEM from 3 biological repeats. Statistics: Two-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ****P < 0.001).