, Cayman Chemicals, 5 µM) as per manufacturer's guidelines and imaged immediately using Zeiss LSM 880 confocal microscope (Carl Zeiss) microscope. and post-fixed with 1% osmium tetroxide and 1% potassium ferrocyanide for 1 h at 4°C, then block stained with 0.01% thiocarbohydrazide following 0.25% aqueous uranyl acetate. The cells were embedded in EMbed, min in distilled water and then stained for 20 min in DHE (12013, vol.812
, Bio-Rad) for SDS-PAGE. Proteins were transferred on polyvinylidene difluoride (PVDF) membrane (BR20160719, Bio-Rad) using a Trans-Blot Turbo Transfer System, mouse anti-mouse HMGB1 (ab11354, Abcam), rabbit anti-mouse phosphoRIPK3 (ab195117, Abcam), rabbit anti-human phosphoRIPK3 (ab209384, Abcam), rabbit anti-mouse/human pDRP1, pp.22989-22990
, Sigma Aldrich) or goat anti-mouse (1:5000 dilution, A0545
Images have been cropped for presentation. Mean band intensity was quantified (ImageJ) and normalized to GAPDH or ?-actin as indicated, were applied before revelation with Clarity Western ECL (#170-5060, Bio-Rad) and imaged on a ChemiDoc Imaging System ,
Concentration of HMGB-1 in mice serum and AT2 cell conditioned medium was determined by an enzyme-linked immunosorbent assay (LS-F23080 ,
, MMP-12 were determined by a MILLIPLEX MAP mouse MMP magnetic bead panel immunology multiplex assay (MMMP3MAG-79K, EMD Millipore) performed according to manufacturer's instructions. A MAGPIX® instrument, MMP multiplex assay. The aortic concentrations of MMP-2, MMP-3, MMP-8, proMMP-9
In vitro transcription was made using clean tailed (120-poly A tail) DNA PCR products. ModRNAs were transcribed in vitro using a custom ribonucleoside blend of Anti Reverse Cap Analog, 30-O-Me-m7G(50) ppp(50)G (6 mM, TriLink Biotechnologies), guanosine triphosphate (1.5 mM, Life Technologies), adenosine triphosphate (7.5 mM, Life Technologies), cytidine triphosphate (7.5 mM, Life Technologies) ,
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are supported by National Institutes of Health (R03-HL142472-01, R01HL146627). F.B. is supported by a Young Investigator Grant from the Alex's Lemonade Stand Foundation ,