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. 2020 Apr 14;10:6343. doi: 10.1038/s41598-020-62875-2

Figure 6.

Figure 6

Effects of MSTN on cell signaling in HK-2 cells. (A) ROS production was measured by DCFH-DA staining and cytofluorimetric reading in HK-2 exposed to MSTN for 5 hours and overnight. Intracellular ROS increased by∼1.5 folds after 5-hour stimulation with MSTN (p < 0.01) and quenched after prolonged exposition (−25% with respect to NC). Values are expressed as fold change ± SEM to no treated cells and are means of 3 independent experiments. (B) Nox 4 expression was evaluated by rt-PCR and immunocytochemistry. Both Nox 4 mRNA and its protein were markedly upregulated (by ∼six folds, p < 0.05 vs. NC) by MSTN. (C) HK-2 were treated for different time intervals (0–60 minutes) with MSTN. Phosphorylated p44/42 MAPKs was detected by Western blot. MSTN induced p44/42 (ERK1/2) phosphorylation (p < 0.01 vs basal value T0).The graph represents relative phospho-p44/42 protein abundance normalized to p44/42 and data are expressed as fold change respect to T0. Immunoblots are representative from 3 different experiments. (D) Effects of PD 98059 (p44/42 MAPK inhibitor) on Nox 4 mRNA in HK-2 exposed to MSTN. Data are expressed as fold change respect to MSTN treated cells and as means of 3 separate experiments. (E) We studied the effects of ERK inhibition by PD 98059 on Nox 4 expression. Nox 4 mRNA was blunted by ERK inhibition (−50% with respect to MSTN-treated cells p < 0.01) suggesting a role of ERK in regulating ROS production by NADPH oxidase. (E) A 48-hour exposition to MSTN promoted phospho-AKT suppression (−40% respect to untreated cells, p < 0.01). The graph depicts phospho-AKT levels normalized to AKT and data are expressed as fold change respect to no treated cells. Immunoblots are representative of 3 different experiments. *p < 0.05, **p < 0.01. Magnification x400. Bar = 35 µM. MSTN = Myostatin; NC = No treated cells; Nox 4 = NADPH oxidase 4; rt-PCR = real-time PCR; MAPK = Mitogen-Activated Protein Kinase; ERK = extracellular signal–regulated kinases; AKT = Protein Kinase B; ROS = Reactive Oxygen Species; DCFH-DA = 2′,7′-Dichlorofluorescin diacetate.