Journal: Nature Communications

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

doi: 10.1038/s41467-024-51232-w

Figure Lengend Snippet: 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.

Article Snippet: The reagents used in this study were listed as follows: Crotonic acid (Sigma-Aldrich, 113018), Sodium acetate (Sigma-Aldrich, S2889), Crotonoyl coenzyme A trilithium salt (Sigma-Aldrich, 28007), Acetyl-coenzyme A trisodium salt (Sigma-Aldrich, 2056), Glyceraldehyde 3-phosphate (Sigma-Aldrich, 39705), β-NAD (Sigma-Aldrich, N8285), ADP (Sigma-Aldrich, A2754), GAPDH (Sigma-Aldrich, G5262), MgCl 2 (Sigma-Aldrich, M8266), Ammonium acetate (Solarbio, A7030), Trichloroacetic acid (Sigma-Aldrich, T6399), Protease inhibitor cocktail (Sigma-Aldrich, 539134), H2DCFDA (MCE, HY-D0940), Mito-tracker (Invitrogen, 22425), Glutathione reduced (Solarbio, G8180), DMEM powder (Caisson, DMP52-10LT), Opti-MEM (Gibco, 31985-070), Puromycin (Gibco, A1113803), Blasticidin (Gibco, A1113903), ECHS1 Fusion protein (Proteintech, Ag16775).

Techniques: Expressing, Silver Staining, Co-Immunoprecipitation Assay, Transfection, Cell Culture, Immunofluorescence, Agarose Gel Electrophoresis, Liquid Chromatography with Mass Spectroscopy, Fractionation, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-51232-w

Figure Lengend Snippet: a The enzymatic activity of Flag-tagged PGK1 WT, K131R, K156R, K220R, or 3KR purified from HEK293T cells were measured. b MDA-MB231 cells expressing Flag-tagged PGK1 WT or 3KR were stimulated with or without hypoxia for 12 h and stained with anti-Flag antibody and Mito-Tracker. Nuclei were stained with DAPI. c Co-IP assays were performed using HEK293T cells transfected with Flag-tagged PGK1 WT or 3KR. d PDHK1 and PDH phosphorylation was detected by immunoblotting in PGK1-deleted MDA-MB231 cells expressing sgRNA-resistant PGK1 (rPGK1) WT or 3KR, with or without shRNA targeting ECHS1. e PDH phosphorylation was detected by immunoblotting in PGK1-deleted MDA-MB231 cells expressing sgRNA-resistant PGK1 (rPGK1) WT or 3KR, with or without PDHK1 depletion. f MDA-MB231 cells with or without ECHS1 depletion were transfected with Flag-tagged PGK1 and cultured under normoxia or hypoxia for 12 h, followed by Co-IP assays. g MDA-MB231 cells with or without ECHS1 depletion were cultured under normoxia or hypoxia for 12 h, followed by immunoblotting with the indicated antibodies. h , i The oxygen consumption rate (OCR, h ) and the extracellular acidification rate (ECAR, i ) in MDA-MB231 cells with endogenous PGK1 deletion and reconstituted expression of rPGK1 WT or rPGK1 3KR, with or without ECHS1 depletion. j Heat map showing the differential metabolites between MDA-MB231/rPGK1 WT cells and MDA-MB231/rPGK1 3KR cells with or without ECHS1 depletion. Each group had 3 biologically independent samples. k The relative levels of [U- 13 C 3 ]-Pyruvate derived m + 2 acetyl-CoA in MDA-MB231/rPGK1 WT cells and MDA-MB231/rPGK1 3KR cells with or without ECHS1 depletion. l The levels of mitochondrial ROS were measured using a 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) probe. a , h – l Data are shown as the mean ± SD, n = 3 ( a , j , k ) or 4 ( h , i , l ) biologically independent samples (unpaired two-tailed Student’s t test). All experimental data were verified in at least two ( j , k ) or three ( a – i , l ) independent experiments. Source data are provided as a Source Data file.

Article Snippet: The reagents used in this study were listed as follows: Crotonic acid (Sigma-Aldrich, 113018), Sodium acetate (Sigma-Aldrich, S2889), Crotonoyl coenzyme A trilithium salt (Sigma-Aldrich, 28007), Acetyl-coenzyme A trisodium salt (Sigma-Aldrich, 2056), Glyceraldehyde 3-phosphate (Sigma-Aldrich, 39705), β-NAD (Sigma-Aldrich, N8285), ADP (Sigma-Aldrich, A2754), GAPDH (Sigma-Aldrich, G5262), MgCl 2 (Sigma-Aldrich, M8266), Ammonium acetate (Solarbio, A7030), Trichloroacetic acid (Sigma-Aldrich, T6399), Protease inhibitor cocktail (Sigma-Aldrich, 539134), H2DCFDA (MCE, HY-D0940), Mito-tracker (Invitrogen, 22425), Glutathione reduced (Solarbio, G8180), DMEM powder (Caisson, DMP52-10LT), Opti-MEM (Gibco, 31985-070), Puromycin (Gibco, A1113803), Blasticidin (Gibco, A1113903), ECHS1 Fusion protein (Proteintech, Ag16775).

Techniques: Activity Assay, Purification, Expressing, Staining, Co-Immunoprecipitation Assay, Transfection, Phospho-proteomics, Western Blot, shRNA, Cell Culture, Derivative Assay, Two Tailed Test

Journal: Nature Communications

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

doi: 10.1038/s41467-024-51232-w

Figure Lengend Snippet: a – g IHC staining ( a ) and the quantitative results ( b – g ) of PGK1 K131cr, PGK1, ECHS1, p-PDHK1, t-PDHK1 and HIF-1α expression in the normal breast tissues from wild-type (FVB) mice and tumor tissues from the MMTV-PyMT mice with early (week 7) or late (week 18) stages of spontaneous breast cancer. Data are shown as the mean ± SD, n = 6 biologically independent samples (unpaired two-tailed Student’s t test). h Expression of PGK1 K131cr, PGK1 and ECHS1 were detected by immunoblotting in samples from the adjacent normal breast tissues (N) and breast tumor tissues (T) using the indicated antibodies. i , j IHC staining ( i ) and the quantitative results ( j ) of PGK1 K131cr expression in samples from adjacent normal breast tissues and breast tumor tissues. Data are shown as the mean ± SD, n (N) = 6 and n (T) = 15 biologically independent samples (unpaired two-tailed Student’s t test). k Kaplan–Meier survival curve showing the overall survival of breast cancer patients according to the levels of PGK1 K131cr expression detected by IHC staining. n (high, 3–4 staining degree) = 70, n (low, 0–2 staining degree) = 59 (log-rank). l PGK1 K131cr expression in breast tumor tissues from patients at different grades (left) or from patients with different lymph node-positive rates (right). m IHC staining of ECHS1 in samples from adjacent normal breast and breast tumor tissues. n Kaplan–Meier survival curve depicting the overall survival of breast cancer patients based on ECHS1 expression in the TCGA cohort. n (high) = 749, n (low) = 287 (log-rank). Source data are provided as a Source Data file.

Article Snippet: The reagents used in this study were listed as follows: Crotonic acid (Sigma-Aldrich, 113018), Sodium acetate (Sigma-Aldrich, S2889), Crotonoyl coenzyme A trilithium salt (Sigma-Aldrich, 28007), Acetyl-coenzyme A trisodium salt (Sigma-Aldrich, 2056), Glyceraldehyde 3-phosphate (Sigma-Aldrich, 39705), β-NAD (Sigma-Aldrich, N8285), ADP (Sigma-Aldrich, A2754), GAPDH (Sigma-Aldrich, G5262), MgCl 2 (Sigma-Aldrich, M8266), Ammonium acetate (Solarbio, A7030), Trichloroacetic acid (Sigma-Aldrich, T6399), Protease inhibitor cocktail (Sigma-Aldrich, 539134), H2DCFDA (MCE, HY-D0940), Mito-tracker (Invitrogen, 22425), Glutathione reduced (Solarbio, G8180), DMEM powder (Caisson, DMP52-10LT), Opti-MEM (Gibco, 31985-070), Puromycin (Gibco, A1113803), Blasticidin (Gibco, A1113903), ECHS1 Fusion protein (Proteintech, Ag16775).

Techniques: Immunohistochemistry, Expressing, Two Tailed Test, Western Blot, Staining