Fig. 3: ECHS1 mediates the downregulation of PGK1 crotonylation under hypoxia. | Nature Communications

Fig. 3: ECHS1 mediates the downregulation of PGK1 crotonylation under hypoxia.

From: Hypoxia-induced downregulation of PGK1 crotonylation promotes tumorigenesis by coordinating glycolysis and the TCA cycle

Fig. 3

a IP assays were performed using HEK293T cells expressing Flag-tagged PGK1, followed by silver staining. b Co-IP assays were performed using HEK293T cells expressing Flag-tagged PGK1. c MDA-MB231 cells were subjected to the Co-IP assays using indicated antibodies. d GST pull-down assays were performed using GST-tagged PGK1, His-tagged ECHS1 or GST proteins. e Co-IP assays were performed using HEK293T cells expressing Flag-tagged PGK1 WT or 3KR. f HEK293T cells were transfected with Flag-tagged PGK1 WT or MUT and subjected to IP assays. g IP assays were performed using HEK293T cells co-expressing Flag-tagged PGK1 WT or 3KR with HA-tagged ECHS1. h MDA-MB231 cells were cultured under normoxia or hypoxia for 12 h and subjected to IP assays. i Immunofluorescence assays were performed using MDA-MB231 cells under normoxia or hypoxia for 12 h. Co-localization was quantified using Pearson’s correlation coefficients. j ECHS1 was detected in MDA-MB231 cells under normoxia or hypoxia for 12 h. k ECHS1 was detected in MDA-MB231 cells expressing scramble or shHIF-1α under normoxia or hypoxia. l ECHS1 mRNA was detected by qPCR in MDA-MB231 cells with or without HIF-1α depletion under normoxia or hypoxia for 12 h. m Agarose-gel-electrophoresis was performed after CUT&Tag assays using HIF-1α antibody in MDA-MB231 cells. The locations of positive #1 and negative #2 primers are shown. n CUT&Tag qPCR assays were performed in MDA-MB231 cells under hypoxia for 12 h using an anti-HIF-1α antibody. o MDA-MB231 cells with or without ECHS1 depletion were cultured under normoxia or hypoxia for 12 h. The levels of crotonyl-CoA in cytosolic and mitochondrial fractions were analyzed by LC-MS/MS. p MDA-MB231 cells were cultured under normoxia or hypoxia for 12 h, followed by fractionation assays. q HEK293T cells with or without ECHS1 depletion were transfected with Flag-tagged PGK1 and subjected to IP assays. In i, l, n, o, data are shown as the mean ± SD, n = 3 (l, n, o) or 6 (i) biologically independent samples (unpaired two-tailed Student’s t test). All experimental data were verified in at least three independent experiments. Source data are provided as a Source Data file.

Back to article page