In vivo evaluation of CXCL6 function in a doxorubicin-induced cardiac toxicity model. (a) Experimental scheme. Full CPC-conditioned medium (CM; from isolate CPC3) was compared with CXCL6-depleted conditioned medium (depCM) for their capacity to counteract DOX toxicity (at 5 mg/Kg). Both CM were injected into the pericardial cavity 6 days after the first DOX dose and compared with mice that received vehicle alone (saline). Four weeks after the initial DOX dose, mice were sacrificed, the heart isolated and analyzed by RT-qPCR or in cardiac sections. Drawings design by Vicente Díaz. (b) ELISA quantification of CXCL6 in CM, depCM and CXCL6 bound to agarose beads. (c,d) All mouse groups were analyzed by RT-qPCR for cardiac expression of myosin light chain 7 (Myl7) and cardiac muscle alpha actin (Actc1) (c), and for Des, Bnp, Casp9, Col1a, Ctgf and Actc1, which are compared with values for mice treated with DOX alone (d). (e) TUNEL evaluation of apoptosis levels in cardiac sections from all mouse groups relative to the negative control. (f) Comparative estimation of collagen I deposition in cardiac sections, after Sirius Red staining. (g) Comparative quantification of angiogenic areas by staining cardiac sections from all groups with anti-CD146 and anti-αSMA: left, representative images scale bar 50 μm; right, quantification in relative units (RU). Data expressed as mean ± SD; black lines indicate p-values (***< 0.002, **< 0.02, *< 0.05; one-way analysis of variance with Bonferroni multiple comparison test).