Fig 1: Constitutive KEAP1 phosphorylation abolishes STK3 deletion-mediated protection against LPS-induced mitochondrial dysfunction and cardiomyocyte death. HL-1 cells were treated with LPS (10 µg/ml) to mimic septic cardiomyopathy in vitro. Non-silencing (control) and STK3-targeted short hairpin RNA (shRNA) was alternatively transfected into HL-1 cells prior to LPS exposure. Phosphodefective (KEAP1T85A) or phosphomimetic (KEAP1T85D) KEAP1 mutants were transfected into cells in the presence of shSTK3. (A, B) Representative images of mitochondrial ROS detection in HL-1 cardiomyocytes. (C-F) ELISA-based analysis of SOD, GSH, GPX, and PRX1 in HL-1 cell homogenates. (G) ELISA-based analysis of caspase-3 activity. (H, I) Assessment of apoptosis in HL-1 cardiomyocytes by TUNEL staining. #p<0.05.
Fig 2: STK3 deletion attenuates LPS-mediated oxidative stress in cardiomyocytes by restoring Nrf2 transcription. HL-1 cells were treated with LPS (10 µg/ml) to mimic septic cardiomyopathy in vitro. Non-silencing (control) and STK3-targeted short hairpin RNA (shRNA) was alternatively transfected into HL-1 cells prior to LPS exposure. Besides, non-silencing control shRNA and Nrf2-targeted shRNA (shNrf2) was alternatively transfected into HL-1 cells prior to LPS exposure. (A, B) Detection of cellular ROS production in HL-1 cells loaded with H2DCFDA. (C, D) Representative images of HL-1 cells loaded with the mitochondrial ROS indicator MitoSox Red. (E-H) ELISA-based analysis of SOD, GSH, GPX, and PRX1 levels in cell homogenates. (I) Analysis of Nrf2 transcriptional expression by qPCR. (J, K) Western blot analysis of Nrf2 expression. #p<0.05.
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