The single protein that causes the fibrosis death spiral NatureComms
cells) was due to the anoikis, a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding ECM. The cells in suspension culture were again cultured on a 10 cm plastic plate . After 7 days culture, the cell number and the viability were measured by Trypan blue staining. Total RNA or protein was collected from the cells.-administered fibrotic liver was perfused with wash buffer [25 mM HEPES/0.
For retroviral transduction of NIH3T3 cells, Plat-E cells were transfected with FLAG-VGLL3 , FLAG-VGLL3 mutant or FLAG-VGLL3 mutant , as shown in Supplementary Table For retroviral transduction of cardiac myofibroblasts or NIH3T3 cells, Plat-E cells were transfected with plasmids with the pMXs-puro backbone, as shown in Supplementary Table, using X-tremeGENE 9 DNA Transfection Reagent. At 48 h after transfection, the supernatant containing the retrovirus was supplemented with 10 μg/mL polybrene and used for transducing the cardiac myofibroblasts or NIH3T3 cells. At 48 h after infection, cells were subjected to immunoprecipitation and immunoblotting.
For immunoblotting to confirm the expression of exogenous VGLL3 and VGLL3 mutants, retrovirally transduced NIH3T3 cells were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors at 4 °C for 20 min. The cell lysates were centrifuged, and the supernatants were mixed with 4× SDS sample buffer and boiled at 95 °C for 5 min.
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