gc 2spd ts cells  (ATCC)

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: The ZCWPW1–ZCWPW2 complex interacts with recombination-associated proteins and stabilizes LDHA activity. ( A ) Schematic of IP-MS-based identification of ZCWPW2-interacting proteins in human testis tissue. ( B ) GO enrichment analysis of ZCWPW2-interacting proteins. ( C ) Mass spectrometry–based intensity plot depicting meiosis-associated proteins that interact with ZCWPW2. ( D and E ) Co-IP analysis of endogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with ZCWPW2 from human (D) and mouse (E) testis lysates. ( F ) Co-IP analysis of exogenous SYCP1, TEX11, HSPA2, MLH1, and MSH2 proteins interacting with exogenous ZCWPW2 in HEK293T cells. ( G–L ) Co-IP assays demonstrating the impact of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (Myc- W1 ) on the interactions of Flag-ZCWPW2 with HA-HSPA2 (G), SYCP1 (H), MLH1 (I), MSH2 (J), TEX11 (K), and MDC1 (L) in HEK293T cells. ( M ) Co-IP assays demonstrating the impact of Zcwpw1 knockdown (siRNA- w1 ) on the interactions of endogenous Zcwpw2 with Hspa2, Sycp1, Mlh1, Msh2, Tex11, Mdc1, and Ldha in GC-2 cells. ( N ) Table summarizing the LDH family proteins detected in ZCWPW2 immunoprecipitates from human testis by IP-MS. ( O ) Co-IP assays demonstrating the interaction between ZCWPW2 and LDHA in human and mouse testes, as well as transfected HEK293T cells. ( P ) Co-IP analysis examining the interaction between Flag-ZCWPW2 and Myc-LDHA in HEK293T cells under conditions of ZCWPW1 knockdown (siRNA- W1 ) or overexpression (HA- W1 ). ( Q ) Schematic overview (top) and quantitative analysis (bottom) of LDH enzymatic activity in testis lysates from WT and Zcwpw2 KO juvenile mice. Activity was measured by assessing formazan absorbance at 450 nm following the conversion of lactate using the WST-8 assay ( n = 3 biologically independent WT mice and KO mice; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( R and S ) LDH enzymatic activity measured by WST-8-based formazan absorbance in treated GC-2 cells (R) and HEK293T cells (S) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Activity Assay, Protein-Protein interactions, Mass Spectrometry, Co-Immunoprecipitation Assay, Knockdown, Over Expression, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A novel dual histone mark reader ZCWPW2 regulates meiotic recombination through lactylation and transcriptional regulation in humans and mice

doi: 10.1093/nar/gkag049

Figure Lengend Snippet: ZCWPW2 promotes lactylation and stability of recombination-associated proteins through coordination with ZCWPW1 in cultured cells. ( A ) Co-IP analysis of HEK293T cells overexpressed with HA-recombination-associated proteins (HSPA2, SYCP1, and MDC1) and Flag-ZCWPW2, with or without Myc-ZCWPW1, and in the presence or absence of siRNA targeting ZCWPW1 . Bar graph quantifies Kla signal intensity for each condition ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( B ) Co-IP analysis (left) for Kla of endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells after individual or combined knockdown of Zcwpw1 or/and Zcwpw2 using siRNAs. Quantification of Kla levels (right) showed a significant decrease in Kla upon Zcwpw1 or Zcwpw2 knockdown, with the greatest reduction observed upon dual silencing ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.). ( C and D ) Co-IP (left) and quantification analysis (right) of Kla on endogenous Mdc1, Hspa2, and Sycp1 in GC-2 cells treated with the EP300 inhibitor A-485, the LDHA inhibitor sodium oxamate, or both (C), and with EP300 activator CTPB or exogenous lactate (D) ( n = 3 independent experiments; two-tailed Student’s t -test; * P < 0.05; error bars, s.e.m.).

Article Snippet: HEK293T cells (CRL-11268; ATCC) and GC-2 cells (CRL-2196; ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS; Gibco, 12483020).

Techniques: Cell Culture, Co-Immunoprecipitation Assay, Two Tailed Test, Knockdown