Journal: Redox Biology
Article Title: Hypobaric hypoxia-driven energy metabolism disturbance facilitates vascular endothelial dysfunction
doi: 10.1016/j.redox.2025.103675
Figure Lengend Snippet: Blocking MPC1 impairs vascular endothelial function though disrupting energy homeostasis. Diagram of cell energy metabolism after blocking MPC1 using siRNA or UK-5099 (10 μM) ( A ); ( B–H ) RAECs was treated with UK-5099 for 24 h to block MPC1. Representative images of immunofluorescence analysis of eNOS ( B ); The protein expression of eNOS, p-eNOS, ZO-1, Claudin-5, Occludin and VE-Cadherin was detected. ∗ p < 0.05 (n = 4) ( C ); The protein expression of MCT4 in RAECs. ∗ p < 0.05 (n = 4) ( D ); Lactate content in the supernatant of RAECs culture. ∗ p < 0.05 (n = 3) ( E ); Lactate content in the cytosol of RAECs, normalized using intracellular protein content. ∗ p < 0.05 (n = 3) ( F ); Representative images of the mitochondrial membrane potential of RAECs detected by JC-10, mitochondrial membrane potential was quantified by red-green fluorescence ratio. ∗∗∗ p < 0.001 (n = 8) ( G ); Representative TEM images of RAECs mitochondria, the arrows indicate mitochondria ( H ); ( I–O ) RAECs were transfected with MPC1 siRNA to knock down MPC1. Representative images of immunofluorescence analysis of eNOS in RAECs after knocking down MPC1 ( I ); The protein expression of eNOS, p-eNOS, ZO-1, Occludin and VE-Cadherin was detected. ∗∗∗ p < 0.001 (n = 4) ( J ); The protein and mRNA levels of MCT4 and MPC1 were detected. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (n = 4) ( K ); Lactate content in the supernatant of RAECs culture. ∗∗ p < 0.01 (n = 3) ( L ); Lactate content in the cytosol of RAECs, normalized using intracellular protein content. ∗ p < 0.05 (n = 3) ( M ); The oxygen consumption rate (OCR) of RAECs after MPC1 knockdown was analyzed by Seahorse XF analyzer, and the ATP production was normalized using intracellular protein content. ∗∗ p < 0.01 (n = 3) ( N ). Representative images of the mitochondrial membrane potential of RAECs detected by JC-10 after knocking down MPC1, and mitochondrial membrane potential was quantified by red-green fluorescence ratio. ∗∗∗ p < 0.001 (n = 7) ( O ).
Article Snippet: Mouse MCT4 (sense: CACGGCAGGUUUCAUAACATT, antisense: UGUUA UGAAACCUGCCGUGTT), MPC1 (sense: GCUAUUCUCUGACAUUCAUTT, antisense: AUGAAUGUCAGAGAAUAGCTT), PKM2 (sense: GCCACAGAAA GCUUUGCAUTT, antisense: AUGCAAAGCUUUCUGUGGCTT) and control small interfering RNA (siRNA) were obtained from GenePharma (Shanghai, China).
Techniques: Blocking Assay, Immunofluorescence, Expressing, Membrane, Fluorescence, Transfection, Knockdown
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