co transfection with pspax2  (Addgene inc)

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: Transfections were performed using jetPRIME® DNA/siRNA (co-) transfection reagent (Polyplus-transfection, Cat No.: 101000001) according to the manufacturer’s instructions.

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) 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: Transfections were performed using jetPRIME® DNA/siRNA (co-) transfection reagent (Polyplus-transfection, Cat No.: 101000001) according to the manufacturer’s instructions.

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