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. 2020 Mar 31;9(4):848. doi: 10.3390/cells9040848

Figure 4.

Figure 4

A-SMase expression regulates mitochondrial function. (A) Measurement of ATP production through oxidative phosphorylation by mitochondria (at 10 min after substrate addition and normalised on the value at time 0) and coupling efficiency of B16-F1_scr and B16-F1_siASM cells (n = 3). * p < 0.05 vs. B16-F1_scr. (B) Left panel: qPCR of HK2 and PFKFB3 on mRNA extract from B16-F1_scr and B16-F1_siASM cells (n ≥ 3). Data are expressed as fold change over B16-F1_scr. Right panel: Measurement of ATP production through glycolysis in B16-F1_scr and B16-F1_siASM cells. Values are expressed as the ATP produced at 10 min after substrate addition and normalised on the value of ATP at time 0 (n = 4). (C) qPCR of CS, MDH, IDH2, IDH3G and SDHA on mRNA extract from B16-F1_scr and B16-F1_siASM cells (n ≥ 3). Data are expressed as fold change over B16-F1_scr. (D) Evaluation by flow cytometry of mitochondrial membrane potential. Left panel: Histograms of TMRM staining of B16-F1_scr and B16-F1_siASM cells. Right panel: Quantification of TMRM staining by measurement of RFI (n = 4). (E) Measurement of ATP production through oxidative phosphorylation by mitochondria of B16-F1_scr and B16-F1_siASM cells treated with Ciplatin (10 μg/mL for 16 h). Values are expressed as the ATP produced at 10 min after substrate addition and normalised on the value of ATP at time 0, n = 3, * p < 0.05 vs. B16-F1_scr. (F) Left panel: Histograms of TMRM staining of B16-F1_scr and B16-F1_siASM cells treated with Cisplatin. Right panel: quantification of TMRM staining by measurement of RFI (n = 4), * p < 0.05 vs. B16-F1_scr.