Fig. 3.
CLC-P/Gal10 inhibits MPM cell response to chemotherapy. (a) Incucyte time-lapse imaging of M14K cells (black) and CFSE-stained Dif-EOL1 cells (green). The white arrow shows a CFSE-labelled granule interacting with M14K cells. (b) Confocal microscopy of primary human eosinophils (Eos) co-cultured with CFSE-labelled M14K cells. After 24 h, cells were fixed, permeabilized, stained with DAPI and labelled with an anti-CD63 APC conjugate (in red). Images were acquired using a Zeiss LSM 880 AiryScan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. White arrows indicate eosinophils. (c) Confocal analysis of Dif-EOL1 cells labelled with DAPI (blue), with an anti-CLC-P/Gal10 antibody and with an AlexaFluor488 conjugate (green). Images were acquired using a Zeiss 880 or 980 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. (d) Representative histogram plot of CLC-P/Gal10 expression acquired by flow cytometry. Dif-EOL1 cells were labelled as described in panel c. (e) Apoptosis of M14K cells in presence of SN Dif-EOL1, SN Dif-EOL1 depleted in CLC-P/Gal10 and/or C + P. CLC-P/Gal10 was depleted from the supernatant by antibody-mediated depletion. Then M14K cells were cultured for 48 h in presence of SN Dif-EOL1 depleted in CLC-P/Gal10. After treatment with C + P for 48 h, cells were labelled with Annexin V/PI and analysed by flow cytometry. (f) Recombinant human CLC-P/Gal10 (0.1, 0.5, 1 and 5 μg/mL) was added in M14K culture medium for 48 h before treatment with C + P. Apoptosis of M14K cells was determined by flow cytometry after Annexin V/PI labelling. (g) Representative images of senescent M14K cells in presence of conditioned medium of differentiated EOL1 (SN Dif-EOL1). M14K cells were cultured for 48 h in presence of mock, SN Dif-EOL1 (25% v/v) or recombinant human CLC-P/GAL10 (1 μg/mL). M14K cells were then treated with 10 μM cisplatin and 10 μM pemetrexed for an additional 2 days. Senescence-associated β-galactosidase (SA-β-gal) activity at pH 6.0 was visualized with an Olympus CKX41 inverted microscope equipped with a 20X objective. (h) The percentages of SA--gal positive cells were counted in ten different fields. Data are expressed as median ±95% CI, each dot representing an independent test. Normality was checked by Shapiro–Wilk and equality of the variances was verified by Brown–Forsythe and Welch. Variance of the means was compared by one-way ANOVA followed by either Tukey's (panels e and f) or Dunnett's T3 (panel h) multiple comparison test.