Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) RNA immunoprecipitation (RIP) from GC-2spc cells using SMG6 and PIWIL1 antibodies. Rabbit IgG was used as a negative control. Left panel shows Western blotting (WB) of SMG6 and PIWIL1. Right panel shows RT-PCR after IP using Ccny and Taf1d specific primers. B) Validation of the knockdown efficiency after transfection of small interfering RNA (siRNA) for Smg6 or Piwil1 (si-Smg6, si-Piwil1) in GC-2spd cells by RT-qPCR using Smg6 or Piwil1 primers. Scrambled siRNA was used as a negative control (si-Scr). C) Validation of the overexpression of SMG6 or PIWIL1 in GC-2spd cells using HA-SMG6 or FLAG-PIWIL1 expression plasmids, respectively. Empty pcdna3.1 vector as a control. D) RT-qPCR using Ccny and Taf1d -specific primers after siRNA-mediated downregulation of Smg6 or Piwil1 in GC-2spd cells. E) RT-qPCR using Ccny and Taf1d -specific primers after overexpression of HA-SMG6 or FLAG-PIWIL1 in GC-2spd cells. F) Overexpression of SMG6 (HA-SMG6) in GC-2spd cells in the presence or absence of Piwil1 knockdown by siRNA. RT-qPCR analysis of Ccny and Taf1d mRNAs was performed to assess whether PIWIL1 is required for SMG6-mediated regulation of these targets. G) Control (si-Scr) or PIWIL1 knockdown (si-Piwil1) GC-2spd cells were subjected to SMG6 RIP, followed by RT-qPCR to assess the association of SMG6 with Ccny and Taf1d mRNAs. Taf1d and Ccny transcripts were quantified and normalized to input RNA. Data represent mean ± S.D. from three independent experiments, *P < 0.05. H) Western blotting with anti-SMG6 antibody to validate equal IP of SMG6 from control (si-Scr) and Piwil1 -silenced (si- Piwil1) cells. Two representative IPs (RIP-1, RIP-2) are shown for both conditions. For all graphs (B-F), means of three independent experiments are shown, and Rpl19 was used as a reference gene. Data represent mean ± S.D. from three independent experiments, *P < 0.05.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: RNA Immunoprecipitation, Negative Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Knockdown, Transfection, Small Interfering RNA, Quantitative RT-PCR, Over Expression, Expressing, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) m⁶A -RIP followed by RT-PCR using primers for Taf1d and Ccny. Mouse IgG was used as a control. B) METTL3 knockdown by siRNA (si-Mettl3) followed by RT-PCR to amplify Ccny and Taf1d mRNAs. Scrambled siRNA (si-Scr) was used as a control. C) Downregulation of METTL3 was validated by anti-METTL3 western blotting. Antibody against ACTIN was used as a loading control. D) GC-2spd cells were treated with the methyltransferase inhibitor STC-15 followed by RT-PCR using Ccny and Taf1d primers (left panel). Right panel shows the levels of m⁶A RNA in control (DMSO) and STC-15-treated cells by dot blotting with anti-m6A antibody (two independent replicates are shown). E) In situ hybridization using Ccny -specific probe of DMSO- or STC-15 treated GC-2spd cells. DAPI stains the nuclei (grey). Scale bar: 10 µm. F) GC-2spd cells were transfected with control siRNA (si-Scr), Smg6 - or Piwil1 -targeting siRNA (si-Smg6 or si-Piwil1), empty pcdna3.1 plasmid (control) or HA-SMG6 and FLAG-PIWIL1 overexpressing plasmids before m⁶A - RIP. m6-A-modified Taf1d was detected with RT-PCR from anti-m⁶A RIP samples. Mouse IgG was used as a control in the RIP. G) GC-2spd cells were treated with DMSO, METTL3a1, or STC-15 before anti-SMG6-RIP. SMG6-associated Ccny and Taf1d mRNAs were detected with RT-PCR. Rabbit IgG was used as a control in the RIP. H) Western blot with anti-SMG6 antibody to validate equal IP of SMG6 from DMSO, STC-15, and METTL3a1 treated cells. One representative RIP experiment is shown for each condition.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Western Blot, In Situ Hybridization, Transfection, Plasmid Preparation, Modification

Journal: bioRxiv

Article Title: Chromatoid body integrates piRNA, SMG6 and m⁶A pathways to control mRNAs in the male germline

doi: 10.64898/2026.03.26.714452

Figure Lengend Snippet: A) Immunoprecipitation of PIWIL1 and SMG6 from adult mouse testes. Successful IP was confirmed with Western blotting (WB) using SMG6 and PIWIL1 antibodies. Associated small RNAs were visualized in the UREA-PAGE gel with SYBR Gold staining, revealing a 26-32-nt population consistent with piRNAs length. B) Transfection of PIWIL1-associated testis RNAs into GC2spd cells, followed by RT-qPCR analysis of Ccny target mRNA. Gapdh was used as a reference gene. Data represent mean ± S.D. from two independent experiments. C) Transfection of Ccny -targeting piRNA ( pi-Ccny ) induces Ccny mRNA downregulation in 3T3 cells only when PIWIL1 is overexpressed. Scrambled piRNA ( pi-Scr ) was used as a control for Ccny -targeting piRNA. Empty pcdna3.1. vector (control) was used as a control for FLAG-PIWIL1 overexpression. D) GC-2spd cells were transfected with pi-Scr or pi-Ccny , followed by RT-qPCR using Ccny primers at different time points after transfection to monitor Ccny mRNA levels. E) GC-2spd cells were subjected to siRNA-mediated knockdown of Smg6 prior to transfection with the synthetic pi-Ccny , followed by RT-PCR analysis of Ccny mRNA. F) Same experiment as in (E), but Ccny expression was detected by RT-qPCR. G) GC-2spd cells were treated with the METTL3 inhibitor STC-15 to block m⁶A deposition, followed by transfection with the synthetic pi-Ccny and RT-PCR analysis of Ccny mRNA. For graphs (D,F), Gapdh was used as a reference gene. Data represent mean ± S.D. from three independent experiments; *P < 0.05.

Article Snippet: Mouse spermatogonial GC-2spd cells (ATCC® CRL-2196TM, USA) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, D-18900; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, S1810; Biowest) and 1% penicillin–streptomycin (15140-122; Gibco).

Techniques: Immunoprecipitation, Western Blot, Staining, Transfection, Quantitative RT-PCR, Control, Plasmid Preparation, Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Blocking Assay

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