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

Journal: Cell reports

Article Title: B cell TET2 recruits OGT for nuclear TET2 and H2B O -GlcNAcylation to drive AID and BLIMP-1 expression and maturation of the antibody response

doi: 10.1016/j.celrep.2026.117370

Figure Lengend Snippet: Naïve IgM + IgD + B cells from Aicda Cre Ogt +/+ and Aidca Cre Ogt fl/fl mice spleen ( n = 3, A–C) or Aicda Cre Tet2 +/+ and Aidca Cre Tet2 fl/fl mice spleen ( n = 3, B and G) were cultured for 72 h or 96 h upon application of GC-like cell-inducing stimuli. (A) Left, CD19 + IgG1 + , CD19 + IgG3 + or CD19 + IgA + B cells and CD19 + CD138 + plasmablasts. Right, intracellular AID and BLIMP-1 expression in B cells stimulated with LPS plus IL-4 after 96 h microculture. (B) Methylated and hydroxymethylated DNA levels in promoter and enhancer regions of Aicda and Prdm1 gene loci in Aicda Cre Tet2 +/+ , Aicda Cre Tet2 fl/fl (top), Aicda Cre Ogt +/+ , and Aicda Cre Ogt fl/fl (bottom) cells after 72 h microculture, as analyzed by MeDIP-qPCR and hMeDIP-qPCR. (C) Expression of TET2 and O -GlcNAcylated TET2 in Aidca Cre Ogt +/+ and Aidca Cre Ogt fl/fl cells after 96 h microculture. (D) Naïve IgM + IgD + B cells from C57BL/6 ( n = 3) mice spleen or healthy humans ( n = 3) were cultured for 0, 24, 48, 72, and 96 h upon stimulation with LPS plus IL-4 or CpG (ODN 2395) plus huIL-2, huIL-4, and huIL-21, respectively. Expression of TET2 and O -GlcNAcylated TET2 in mouse and human B cells at the indicated time points. (E) TET2 O -GlcNAcylation sites in mouse B cells, as predicted by YinOYang and O -GlcNAcPRED-DL. (F) Naïve IgM + IgD + B cells from C57BL/6 mice ( n = 3) spleen were cultured for 72 h upon stimulation with LPS plus IL-4. TET2 or OGT interaction with TET2, OGT, PROSER1, HDAC1, and CREBBP in cell nuclei, as analyzed by specific immunoblots. (G) Aidca Cre Tet2 +/+ and Aidca Cre Tet2 fl/fl B cell OGT (left) and O -GlcNAc H2B-S112 (right) levels after 72 h microculture, as expressed in promoter and enhancer regions of Aicda and Prdm1 gene loci, as analyzed by ChIP-qPCR. Statistical significance (B and G) was assessed using two-sided Student’s unpaired t test (** p < 0.01, *** p < 0.001).

Article Snippet: anti-mo/hu-CREBBP mAb , ABclonal , Cat. # A26780.

Techniques: Cell Culture, Expressing, Methylation, Methylated DNA Immunoprecipitation, Western Blot, ChIP-qPCR