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In addition, it has been shown that FGF2 can be internalized via FGFR1-dependent and FGFR1-independent (Syndecan-4) pathways (Tkachenko et al

In addition, it has been shown that FGF2 can be internalized via FGFR1-dependent and FGFR1-independent (Syndecan-4) pathways (Tkachenko et al. (PNG 637?kb) 441_2018_2905_Fig7_ESM.png (637K) GUID:?4789CE0A-91F5-4D48-A0CF-71C9C71D4CF3 High Resolution (TIF 25.6 mb) 441_2018_2905_MOESM1_ESM.tif (26M) GUID:?5CD3D8D3-75B5-4E2A-8E0B-F04A20F3CB37 Figure S2: MK-5172 sodium salt Neonatal rat cardiomyocytes were stimulated with S117A-FGF2 or FGF2 (10?ng/ml) for 60?min. Both S117A-FGF2 and FGF2 were able to increase phosphorylation of AKT (tyrosine 473) and P38 (panels a-a). Phospho-ERK was also upregulated at this time point, included for comparisons. The experiment was done with for 15?min) to remove debris. Bicinchoninic acid (BCA) assay was used to determine protein concentration in the samples. Following SDS-PAGE, proteins were transferred to polyvinylidene fluoride (PVDF) membranes. 0.01% (value was set at em P /em ? ?0.05. The data are shown as mean standard error mean (SEM). Results The effect of S117A-FGF2 against Dox-induced cardiomyocyte damage We recently reported that primary cultures of neonatal rat cardiomyocytes, referred to as cardiomyocytes from now on, represent a good in vitro model to study multiple aspects of acute Dox-induced toxicity (Koleini et al. 2017). This model was used to examine the effect of pretreatment with S117A-FGF2 and FGF2 (used as positive control), on Dox-induced LDH release and cell death. As shown in Fig. ?Fig.1(a),1(a), S117A-FGF2 was equally able as FGF2 in reducing cardiomyocyte plasma membrane damage compared to Dox, estimated by decreased LDH release. Using a Calcein-AM/ethidium MK-5172 sodium salt homodimer assay, where live cells stain green (calcein-AM) and lifeless cells are stained red (ethidium homodimer), S117A-FGF2 was found to decrease the relative abundance of lifeless cells significantly Fig. ?Fig.1(b,1(b, cCc). Open in a separate windows Fig. 1 Non-mitogenic (S117A) FGF2 protects cardiomyocytes from Dox toxicity. (a) The effect of doxorubicin (Dox) on LDH released to the medium by MK-5172 sodium salt cardiomyocytes, as well as the effect of pretreatment with S117A-FGF2, versus FGF2, on Dox-induced LDH release, as indicated. (b) The effect of Dox around the percentage of lifeless cells, as measured by the calcein-AM/ethidium homodimer assay; the effect of pretreatment with S117A-FGF2, or FGF2, on Dox-induced cell death is also shown. Representative fluorescence images are included in (c-c), where green or red stain live or lifeless cells, respectively. Brackets mark groups that differ significantly from each other ( em n /em ?=?4), in both (a) and (b) We next examined the effect of S117A-FGF2 on a breast malignancy cell line, MCF-7 cells. MCF-7 exposure to Dox for 24?h increased LDH release, an effect that was not prevented by S117A-FGF pre-incubation (Supplement, Fig. S1). Using an MTT assay as an estimate of cell number, we found that S117A-FGF2 had no effect on proliferation, unlike FGF2 that elicited a small but statistically significant increase in cell number (Fig. S1). The role of FGFR1 and ERK in S117A protection against dox-induced cardiomyocyte damage Fibroblast growth factor receptor-1 (FGFR1) KIR2DL5B antibody is the main cardiomyocyte FGF2 receptor (Kardami et al. 1995; Liu et al. 1995). Upon ligand binding, FGFR1 dimerizes and becomes trans-phosphorylated on tyrosine (Y) 766 (Ornitz and Itoh 2015). As shown in Fig.?2(a, a), S117A-FGF2 stimulation for 30?min significantly upregulated relative levels of pY766-FGFR1 compared to unstimulated cells. Increased pY766-FGFR1 levels in S117A-FGF2-treated MK-5172 sodium salt cells persisted even after Dox exposure, Fig. ?Fig.2(a,2(a, a). Pre-incubation of cardiomyocytes with the specific FGFR1 inhibitor PD173074 abolished the S117A-FGF2-induced protection from Dox-induced LDH release (Fig. ?(Fig.22b). MK-5172 sodium salt Open in a separate windows Fig. 2 S117A-FGF2 requires FGFR1 to protect cardiomyocytes against Dox damage. (a) The effect of S117A-FGF2 on relative pY766-FGFR1 after 30-min cardiomyocyte stimulation and after Dox exposure for another 30?min. Brackets show groups significantly different from each other ( em n /em ?=?3). Images of the corresponding western blot probed for anti-pY766-FGFR1, or stained with Ponceau S are also included (a). (b) The effect of PD173074 (FGFR1 inhibitor) on S117A-FGF2 protection from Dox-induced LDH release. Brackets point to groups that are significantly different from each other. In the presence of PD173074, S117A-FGF2 is unable to reduce Dox-induced LDH release Several kinases associated with regulation of cardioprotection are known to be activated downstream of the FGF2/FGFR1 axis. These include ERK (or p44/42 MAPK), p38 and AKT (Katoh 2016). Exposure of cardiomyocytes to S117A-FGF2 for 30?min upregulated P-ERK, compared to unstimulated cells (Fig.?3a, a). Phospho-ERK remained elevated in S117A-FGF2-treated cells even after incubation with Dox for 30?min (Fig. ?(Fig.3a,3a, a). The specific ERK inhibitor U0261 abolished S117A-FGF mediated cardiomyocyte protection against Dox (Fig. ?(Fig.3b).3b). In addition, as shown in Fig. S2, both mitogenic FGF2 and S117-FGF2 upregulated cardiomyocyte P-p38 as well as P-AKT, after 60?min. Open in a separate windows Fig. 3 The S117A-FGF2 protection from Dox requires ERK activity. (a) The effect of S117A-FGF2 on pERK/ERK ratio after 30 min cardiomyocyte stimulation and after Dox exposure for another 30?min. Images.