Journal: Clinical and Translational Medicine

Article Title: ROS‐ATM‐CBP axis‐mediated PARP1 lactylation aggravates doxorubicin‐induced cardiotoxicity

doi: 10.1002/ctm2.70745

Figure Lengend Snippet: CBP is the lactyltransferase for PARP1 lactylation. (A, B) Lactylation of endogenous PARP1 was determined by Co‐IP using anti‐PARP1 or anti‐PanKla antibodies in HL‐1 cells. (C) CBP can specifically catalyze lactylation of PARP1 in vitro. (D, E) Co‐IP of endogenous proteins from HL‐1 cell lysates using anti‐CBP or Anti‐PARP1 antibody, followed by immunoblot of CBP, PARP1 and Tubulin. (F, G) HL‐1 cells treated with or without CBP activator or CBP inhibitors were lysed and immunoprecipitated using anti‐PARP1 antibody, followed by detection of Pan‐KLA. (H) Full‐length Myc‐PARP1 or six truncated Myc‐PARP1 plasmids were transfected, respectively, and total lysates were subjected to immunoprecipitation with anti‐Myc antibody, followed by detection of Pan‐KLA. (I) The mass spectrometry analysis results of the lactylation site of PARP1. (J) HL‐1 cells transfected with Myc‐PARP1‐WT, Myc‐PARP1‐mut 5KR and Flag‐CBP were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. (K) HL‐1 cells transfected with Myc‐PARP1‐WT and Myc‐PARP1‐mut 5KR, with or without the treatment of CBP inhibitor, were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. All immunoblots had three biological repetitions, with similar results.

Article Snippet: ATM inhibitor (KU‐55933; #HY‐12016; 10 μM; 2 h); LDHA inhibitor (Galloflavin; #HY‐W040118; 10 −5 M; 24 h) and CBP activator (CTB; #HY‐134964; 10 −4 M; 24 h) were obtained from MedChemExpress.

Techniques: Co-Immunoprecipitation Assay, In Vitro, Western Blot, Immunoprecipitation, Transfection, Mass Spectrometry

Journal: Clinical and Translational Medicine

Article Title: ROS‐ATM‐CBP axis‐mediated PARP1 lactylation aggravates doxorubicin‐induced cardiotoxicity

doi: 10.1002/ctm2.70745

Figure Lengend Snippet: ROS‐ATM‐CBP signalling pathway activates PARP1 lactylation. (A, B) Intracellular ROS and lactate levels in HL‐1 cells treated with 1 µM doxorubicin for 0, 2 and 4 h. (C) Representative immunoprecipitation, western blots and quantification for detecting PARP1 lactylation. HL‐1 cells were stimulated by doxorubicin (1 µM for 0, 2 and 4 h), and the cells were lysed and immunoprecipitated using an anti‐PARP1 antibody, followed by detection of Pan‐KLA. (D) Schematic of the effect of SIRT1 activation on the lactylation level of PARP1 in mice. DOX (5 mg/kg) or the same volume of saline was administered intraperitoneally once weekly. One week before the DOX treatment, mice were pre‐treated with resveratrol (SIRT1 activator, 10 mg/kg/day) via daily intraperitoneal injections. (E) The SIRT1 activity was detected. (F–H) Representative immunoprecipitation, western blots and quantification for detecting PARP1 lactylation and protein expression of SIRT1. (I) Schematic of the effect of CBP inhibition on the lactylation level of PARP1 in mice. DOX (5 mg/kg) or the same volume of saline was administered intraperitoneally once weekly. One week before the DOX treatment, mice were pre‐treated with SGC‐CBP30 (CBP inhibitor, 15 mg/kg/day) via daily intraperitoneal injections. (J) The CBP activity was detected. (K–M) Representative immunoprecipitation, western blots and quantification for detecting PARP1 lactylation and protein expression of CBP. (A–C) Based on the central limit theorem, the data were considered to be normally distributed. Relative protein levels were calculated as fold changes vs the first group. Data are presented as mean ± SD. Statistical significance was assessed by one‐way ANOVA with Tukey multiple comparisons test ( P values adjusted for 3 comparisons). (D–M) Relative protein levels were calculated as fold changes vs. the first group. Data are presented as mean ± SD. Statistical significance was assessed by two‐way ANOVA with Bonferroni multiple comparisons test ( p ‐values adjusted for 9 comparisons). ns p > .05, * p < .05, ** p < .01, *** p < .001.

Article Snippet: ATM inhibitor (KU‐55933; #HY‐12016; 10 μM; 2 h); LDHA inhibitor (Galloflavin; #HY‐W040118; 10 −5 M; 24 h) and CBP activator (CTB; #HY‐134964; 10 −4 M; 24 h) were obtained from MedChemExpress.

Techniques: Immunoprecipitation, Western Blot, Activation Assay, Saline, Activity Assay, Expressing, Inhibition