piwil2  (Proteintech)

Journal: Biomedicines

Article Title: Atrazine Induces Reproductive Toxicity in an In Vitro Spermatogenesis (IVS) Model

doi: 10.3390/biomedicines13122917

Figure Lengend Snippet: Directed differentiation confirmation of WT and HD-hiPSCs into SSCLCs in vitro . ( a ) Schematic representation of the ATZ exposure and SSCLC differentiation model from hiPSCs (with and without ATZ treatment). ( b ) Bright-field images at day 0 and day 15 of differentiation (scale bar = 100 μm). ( c ) ICC images at day 15 showing expression of PIWIL2 and ZBTB16 in all cell lines (scale bar = 100 μm). ( d ) qRT-PCR analysis showing relative gene expression profiles after SSCLC differentiation. Both WT and HD cell lines exhibited a significant decrease in pluripotency markers ( OCT4 and SOX2 ) and an upregulation of SSC markers such as DAZL , ZBTB16 , GFRA1 , and PIWIL2 from day 5 to day 15 of differentiation. Data are presented as mean ± SEM from three biological replicates (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. Comparisons were made with the hiPSC state of each cell line: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Then cells were permeabilized with 0.2% Triton X-100 solution for 15 min and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) and 5% normal goat serum (NGS) for 1 h. Cells were sequentially incubated overnight at 4 °C with primary antibodies against OCT4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), SOX2 (1:500; STEMCELL Technologies), PLZF/ZBTB16 (1:200; R&D Systems), and PIWIL2 (1:200; Santa Cruz Biotechnology).

Techniques: In Vitro, Expressing, Quantitative RT-PCR, Gene Expression, Comparison

Journal: Biomedicines

Article Title: Atrazine Induces Reproductive Toxicity in an In Vitro Spermatogenesis (IVS) Model

doi: 10.3390/biomedicines13122917

Figure Lengend Snippet: Comparison of relative SSC gene expression profiles between WT and HD hiPSCs during differentiation into SSCLCs. qRT-PCR analysis shows that, under ATZ untreated conditions, SSC gene expression profiles after SSCLC differentiation did not differ significantly between WT and HD lines. Both cell types exhibited upregulation of SSC markers, including DAZL , ZBTB16 , GFRA1 , and PIWIL2 , during the transition from hiPSCs to SSCLCs. Comparisons were made with the hiPSC state of each cell line. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then cells were permeabilized with 0.2% Triton X-100 solution for 15 min and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) and 5% normal goat serum (NGS) for 1 h. Cells were sequentially incubated overnight at 4 °C with primary antibodies against OCT4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), SOX2 (1:500; STEMCELL Technologies), PLZF/ZBTB16 (1:200; R&D Systems), and PIWIL2 (1:200; Santa Cruz Biotechnology).

Techniques: Comparison, Gene Expression, Quantitative RT-PCR

Journal: Biomedicines

Article Title: Atrazine Induces Reproductive Toxicity in an In Vitro Spermatogenesis (IVS) Model

doi: 10.3390/biomedicines13122917

Figure Lengend Snippet: Effect of ATZ exposure during human spermatogenesis in vitro . ( a – c ) qRT-PCR analysis of relative gene expression of early SSC ( DAZL , ZBTB16 , and GFRA1 ) and late SSC ( PIWIL2 ) markers at day 5 and day 15 of SSCLC differentiation in WT, HD1 and HD2, respectively. ( d ) Summary plot of SSC related gene expression at day 5 of SSCLC differentiation in WT, HD1 and HD2 compared to untreated control, respectively. ( e ) Summary plot of SSC related gene expression at day 15 of SSCLC differentiation in WT, HD1 and HD2, respectively. Gene expression is shown using a pseudo–color scale from red to blue. Red squares indicate significant upregulation, blue squares indicate significant downregulation, and gray squares indicate no significant difference ( p > 0.05). Data are presented as mean ± SEM from three biological replicates (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Then cells were permeabilized with 0.2% Triton X-100 solution for 15 min and blocked with 2% bovine serum albumin (BSA; Sigma-Aldrich) and 5% normal goat serum (NGS) for 1 h. Cells were sequentially incubated overnight at 4 °C with primary antibodies against OCT4 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), SOX2 (1:500; STEMCELL Technologies), PLZF/ZBTB16 (1:200; R&D Systems), and PIWIL2 (1:200; Santa Cruz Biotechnology).

Techniques: In Vitro, Quantitative RT-PCR, Gene Expression, Control, Comparison