Fig. 4.
CLC-P/Gal10 expressed by primary eosinophils impairs the cytotoxic activity of cisplatin and pemetrexed. (a) Schematic representation of the experimental protocol for the isolation of primary human eosinophils. Primary eosinophils were purified from the polymorphonuclear cell (granulocytes)-rich fraction of peripheral blood by Ficoll gradient centrifugation and positively selected by magnetic-activated cell sorting using an anti-CCR3 antibody. The culture supernatant of CCR3-positive eosinophils (SN Eos) was collected after 24 h and added at a ratio of 25% (v/v) to M14K cells for 48 h. Then, M14K cells were treated with C + P for 48 h and analysed by flow cytometry after Annexin V/PI labelling. (b) Purified CCR3+ eosinophils were fixed, permeabilized and labelled with DAPI (blue), CLC-P/Gal10 (green) and MBP (red). Images were acquired using a Zeiss 980 Airyscan Elyra confocal microscope equipped with a x63–1.4 oil immersion objective. (c) Representative histogram plot of CLC-P/Gal10 expression acquired by flow cytometry. Primary eosinophils were labelled with an anti-CLC-P/Gal10 antibody and an AlexaFluor488 conjugate. (d) Apoptosis of M14K cells in presence of SN Eos and/or C + P. M14K cells were cultured with SN Eos for 48 h. After treatment with C + P for 48 h, cells were labelled with Annexin V/PI and analysed by flow cytometry. (e) Same as in panel d except that CLC-P/Gal10 protein was depleted from SN Eos by using an anti-Gal10 antibody. (f) Representative images of senescent M14K cells in presence of SN Eos. M14K cells were cultured for 48 h in presence of SN Eos treatment with 10 μM cisplatin and 10 μM pemetrexed for an additional 2 days. Senescence-associated -galactosidase (SA-β-gal) activity at pH 6.0 was visualized with an Olympus CKX41 inverted microscope equipped with a 20X objective. (g) The percentages of SA-β-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 were determined by Brown–Forsythe and Welch. Variance of the means was compared by one-way ANOVA followed by Tukey's (panel e) or Dunnett's T3 (Panels d and g) multiple comparison test. (h) Graphical summary of conclusions drawn from cell culture experiments.