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. 2016 Jul 21;6:29994. doi: 10.1038/srep29994

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Copyright © 2016, Macmillan Publishers Limited

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(a) Western blot analysis for the expression of phosphorylated IκBα (Ser^32/36 p-IκBα, 40kDa) in untreated mNVCM or mNVCM treated with 1 μM Dox, with 40 μg/ml hAFS-CM_Hypo prior to exposure to 1 μM Dox, or with 20 μM LY-294002 (PI3K inhibitor) followed by 40 μg/ml hAFS-CM_Hypo and 1 μM Dox. Full-length blots are represented in Supplementary Fig. S4. (b) ELISA analysis for nuclear expression of the p65 subunit of the NF-kB complex in untreated mNVCM (Ctrl) or mNVCM treated with 1 μM Dox (Dox), with 40 μg/ml hAFS-CM_Hypo followed by 1 μM Dox (hAFS-CM_Hypo + Dox), or with 40 μg/ml hAFS-CM_Hypo followed by 1 μM Dox after pre-incubation with 20 μM LY-294002 (LY-294002 + hAFS-CM_Hypo + Dox). Pg: picogram; μg: microgram. (c) Real time qRT-PCR showing that treatment of mNVCM with 20 μM LY-294002 (LY-294002 + hAFS-CM_Hypo + Dox) also abrogated the upregulation of Il6 and decreased that of Cxcl1 attained by pre-incubation with 40 μg/ml hAFS-CM_Hypo prior to exposure to 1 μM Dox (hAFS-CM_Hypo + Dox). *p < 0.05 (p = 0.0309 and p = 0.0324, for Il6 and Cxcl1 respectively), **p < 0.01 (p = 0.0015), ***p < 0.001 (p = 0.0006). (d) Percentage of mNVCM expressing SA β-galactosidase (upper panel) and cleaved caspase-3 (% Caspase-3⁺ cells, lower panel): untreated cells (Ctrl), cells exposed to 1 μM Dox (Dox), and cells treated with 40 μg/ml hAFS-CM_Hypo followed by 1 μM Dox (hAFS-CM_Hypo + Dox) or with 40 μg/ml hAFS-CM_Hypo followed by 1 μM Dox after pre-incubation with 20 μM LY-294002 (LY-294002 + hAFS-CM_Hypo + Dox). Note the reversion of the cardioprotective effects of the hAFS secretome with LY-294002 (LY-294002 + hAFS-CM_Hypo + Dox: 46.37 ± 6.84% of SA β-galactosidase positive cells and 33.80 ± 0.94% of cleaved caspase-3 positive cells, values expressed as mean ± s.e.m.; *p < 0.05 (p = 0.0208), ****p < 0.0001. (e) Western blot analysis (left panel) and corresponding densitometry (right panel) for phosphorylated Akt (Ser⁴⁷³ p-Akt, 60kDa) in untreated mNVCM (Ctrl) or mNVCM treated with 1 μM Dox (Dox), with 40 μg/ml hAFS-CM_Hypo (hAFS-CM_Hypo), or with 20 μM LY-294002 (PI3K inhibitor) followed by 40 μg/ml hAFS-CM_Hypo (LY-294002 + hAFS-CM_Hypo). Full-length blots are represented in Supplementary Fig. S4. **p < 0.01 (p = 0.0011), ****p < 0.0001.