Figure 3. The pro-survival genes Il6 and Cxcl1 are up-regulated by incubation with the hAFS-CM_Hypo prior to Dox exposure and play a pivotal role in hAFS-CM_Hypo cardioprotection.

(a) Schematic illustrating the cluster of genes coding for cytokines, chemokines, growth factors, and chemokine and cytokine receptor-binding proteins up-regulated by incubation with 40 μg/ml of hAFS-CM_Hypo prior to exposure to 1 μΜ Dox, according to microarray analysis. (b) Real time qRT-PCR showing significant up-regulation of the NF-κB-controlled pro-survival genes Il6 and Cxcl1 in mNVCM incubated with 40ug/ml of hAFS-CM_Hypo prior to treatment with 1 μM Dox; **p < 0.01 (p = 0.0015 and p = 0.0020, respectively). (c) Percentage of mNVCM expressing cleaved caspase-3 (% Caspase-3⁺ cells) after exposure to Dox, with or without pre-incubation with 40 μg/ml of the hAFS-CM_Hypo and with blocking antibodies against IL-6 (anti-IL6) and/or CXCL-1 (anti-CXCL1) added to the culture medium (mean ± s.e.m.). Ctrl: 18.55 ± 4.0%, Dox: 42.57 ± 7.7%, hAFS-CM_Hypo + Dox: 25.25 ± 3.5%, The following values refer to results obtained adding blocking antibodies when Dox treatment began, after 3 hours of incubation with the hAFS-CM: hAFS-CM_Hypo + Dox + anti-IL-6: 42.79 ± 2.7%, hAFS-CM_Hypo + Dox + anti-CXCL-1: 37.94 ± 1.8%, hAFS-CM_Hypo + Dox + anti-IL-6 and+ anti-CXCL-1: 39.68 ± 9.1%, ****p < 0.0001, ***p < 0.001 (p = 0.0002), **p < 0.01 (p = 0.0041) *p < 0.05 (p = 0.0181). The following values refer to results obtained adding blocking antibodies 6 and 9 hours after Dox treatment and hAFS-CM incubation, respectively: hAFS-CM_Hypo + Dox + anti-IL-6: 41.75 ± 1.1%, hAFS-CM_Hypo + Dox + anti-CXCL-1: 34.83 ± 7.1%, hAFS-CM_Hypo + Dox + anti-IL-6 + anti-CXCL-1: 41.75 ± 5.1%; ***p < 0.001 (p = 0.0006).