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. 2020 Mar 17;21(6):2064. doi: 10.3390/ijms21062064

Figure 10.

Figure 10

Neutralizing S1P with S1P-antibody attenuates TGF-β- and S1P-mediated mtROS in HLFs. Human lung fibroblasts (HLFs) grown on 35 mm glass-bottom dishes were pre-treated with sphingosine-1-phosphate (S1P) antibody (25 µg/mL) for 1 h prior to TGF-β (5 ng/mL) (A,B) or S1P (1 µM) (C,D) challenge for 24 h. After 24 h, cells were treated with MitoSOX for 30 min as described in Methods, and mitochondrial ROS (mtROS) was quantified using confocal microscopy. Cells (20–30 numbers) from at least 5–8 different areas of three independent experiments were used for quantification. ** p < 0.001, and *** p < 0.0001 as compared to controls. S1P antibody attenuated both TGF-β- and S1P-induced mtROS in HLFs, showing the role of its extracellular signaling via its receptors. Scale bar: 20 μm.