Journal: The EMBO Journal
Article Title: SUCLG1 restricts POLRMT succinylation to enhance mitochondrial biogenesis and leukemia progression
doi: 10.1038/s44318-024-00101-9
Figure Lengend Snippet: ( A – C ) ETC correlation scores of succinyl-CoA-metabolizing enzymes, SUCLA2 ( A ), SUCLG2 ( B ), and OGDH ( C ), were determined in various TCGA cancers. Shown are Spearman’s correlation coefficients. ( D ) OGDH protein expression in stable HL60 and MV411 cells was determined by western blotting. ( E ) Extracellular acidification rates of control or SUCLG1 -knockdown HL60 cells were determined. Data represent means ± SD, n = 3 independent biological replicates, t test. ** P < 0.01; n.s. not significant. ( F , G ) Glucose (Glc) consumption and lactate production were assayed in control and SUCLG1 -knockdown HL60 cells. Data represent means ± SD, n = 3 independent biological replicates, t test. ** P < 0.01. ( H ) ATP abundance was quantified in control and SUCLG1 -knockdown HL60 cells, and normalized to total protein. Data represent means ± SD, n = 3 independent biological replicates, t test. ** P < 0.01; * P < 0.05. ( I ) Schematic overview of metabolic enzymes and transporters involved in succinate metabolism. ( J ) Mitochondria were isolated from stable SUCLG1 -knockdown and rescue MV411 cells. Whole-cell lysate and mitochondrial fractions were subject to western blotting. Histone H3, G6PD, and HSP60 were included as markers for the nucleus, cytosol, and mitochondria, respectively. ( K ) HA-tagged POLRMT was expressed in scrambled control or SUCLG1 -knockdown MV411 cells. POLRMT-HA was immunoprecipitated from stable MV411 cells and subjected to western blotting to determine lysine succinylation (suc-Lys), lysine methylation (me-Lys), lysine acetylation (ac-Lys), and tyrosine phosphorylation (p-Tyr). ( L ) Myc-tagged POLRMT and HA-tagged ubiquitin were co-expressed in stable MV411 cells. POLRMT was immunopurified with Myc antibody to determine its ubiquitination by western blotting. ( M ) Endogenous POLRMT was immunoprecipitated from control or SUCLG1 -knockdown MV411 cells. Ubiquitination was determined by western blotting. ( N ) Bacterially expressed His-tagged POLRMT was purified and incubated with succinyl-CoA (sucCoA) or succinate in vitro. Lysine succinylation levels were determined by western blotting and normalized to POLRMT-His protein (ratio). ( O ) Scrambled control or SUCLG1 -knockdown (sh-#1) MV411 cells were cultured with 1 mM pyruvate or 0.2 mM uridine. Colony formation was determined. Data represent means ± SD, n = 3 independent biological replicates, t test. ** P < 0.01; n.s. not significant. .
Article Snippet: Antibodies against MT-CO1 (Novus biologicals #NBP2-29949), MT-ND6 (Novus biologicals #NBP1-70650), MT-ATP6 (Sigma #MABS1995), SDHA (Cell Signaling #11998), NDUFB8 (Novus biologicals #NBP2-75586), POLG (Abcam #ab128899), POLRMT (Abcam #ab167368), MRPL45 (Sigma #HPA023373), PGC-1α (Abcam #ab54481), NRF2 (Abcam #ab89443), beta-actin (Cell Signaling #3700), Flag (Sigma #F1804), SUCLG1 (Abcam #ab97867), TFAM (Cell Signaling #8076), suc-Lys (Novus biologicals #NBP3-16032), HA (Santa Cruz #sc-7392), HSP60 (Cell Signaling #4870), G6PD (Abcam #ab133525), Histone H3 (Cell Signaling #3638), me-Lys (Cell Signaling #14679), ac-Lys (Cell Signaling #9441), p-Tyr (Santa Cruz #sc-508), SUCLG2 (Santa Cruz #sc-390818), SUCLA2 (Novus biologicals #NBP1-33015), His (Cell Signaling #2366), TFB2M (Cell Signaling #50837), FLT3 (Cell Signaling #3462), Myc tag (Cell Signaling # 2276), OGDH (Abcam #ab137773), Ubiquitin (Abcam #ab134953), SDHB (Abcam #ab175225), FH (Abcam #ab233394), SIRT5 (Abcam #ab259967), POLG (Abcam #ab128899), E2F1 (Santa Cruz #sc-251) were purchased commercially.
Techniques: Expressing, Western Blot, Control, Knockdown, Isolation, Immunoprecipitation, Methylation, Purification, Incubation, In Vitro, Cell Culture