Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: CRISPR/Cas9 induces DNA damage and apoptosis in PGCs. (A) Flow cytometry analysis 24 h after electroporation, quantifying the proportion of Annexin V + /PI + cells. The horizontal axis indicates PI and the vertical axis Annexin V. The upper-left quadrant (Annexin V + /PI + ) represents late apoptotic cells, and the lower-right quadrant (Annexin V + only) represents early apoptotic cells. Upper panels: results after electroporation with Cas9 + various gRNAs; lower panels: results with dCas9 + various gRNAs. (B) Bar graph of Annexin V + /PI + percentages across groups. Cas9 editing induced a highly significant increase in late apoptosis. (C) γ-H 2 AX foci (green) detected by immunofluorescence 24 h after electroporation. Foci appear as discrete nuclear puncta; nuclei are counterstained with DAPI (blue). Scale bar = 10 µm. (D) Quantification of γ-H 2 AX foci per cell. Cas9 targeting resulted in a significant increase in γ-H 2 AX foci per cell, whereas dCas9 with sgRNA did not. Statistical significance determined by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: CRISPR, Flow Cytometry, Electroporation, Immunofluorescence

Journal: Poultry Science

Article Title: High genotoxicity of CRISPR/Cas9 versus limited efficacy of CRISPRi in chicken primordial germ cells

doi: 10.1016/j.psj.2026.106722

Figure Lengend Snippet: PGCs are more sensitive to DNA double-strand breaks than somatic cells. (A) Cell viability curves after 24 h treatment with increasing concentrations of etoposide (ETP; x-axis shows log 10 [ETP]). PGC viability dropped sharply even at low ETP doses, whereas CEF cells were more tolerant (viability data are mean ± SD of triplicates). (B) Apoptosis detection in PGCs treated with low-dose ETP. The proportion of Annexin V + /PI + cells (late apoptosis) was significantly elevated even at 0.03 µM ETP. (C) Western blot analysis of γ-H 2 AX protein in PGCs treated with varying ETP concentrations (left panel; β-actin as loading control). The bar graph (right) shows the ratio of γ-H 2 AX to β-actin band intensity, with a marked increase at 3 µM ETP (p-values are indicated in the figure). (D) Quantification of γ-H 2 AX foci per cell in THP-1 cells, male PGCs, and female PGCs after exposure to X-ray doses of 0, 2, 4, 6, and 8 Gy. After 48 h recovery, γ-H 2 AX foci increased significantly with higher radiation in THP-1, male PGCs, and female PGCs. Female PGC data points are red squares; male PGCs are blue squares; THP-1 are black circles. (E) Cell-cycle distribution of male vs. female PGCs after DNA damage. PGCs were irradiated (2, 4, 6 Gy), cultured 48 h, and analyzed by flow cytometry for cell-cycle phase (propidium iodide staining). Stacked bars show the percentage of cells in G 0 G 1 , S, and G 2 /M phases in untreated vs. irradiated cells. After damage, female PGCs predominantly arrested in G 2 /M (increased G 2 fraction), whereas male PGCs accumulated in S phase. Statistical significance by one-way ANOVA (p-values are indicated in the figure).

Article Snippet: After another centrifugation, the supernatant was removed and cells were resuspended in 500 μL of 1× Annexin V Binding Buffer (from Annexin V-FITC/PI Apoptosis Kit, Elabscience E-CK-A211).

Techniques: Western Blot, Control, Irradiation, Cell Culture, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Clinical-grade cryopreservation unlocks transplant-ready human pancreatic and stem cell–derived islets for diabetes therapy

doi: 10.64898/2026.04.25.720819

Figure Lengend Snippet: ( A, B ) Bulk RNA-seq volcano plots comparing fresh versus VR native islets ( A ) and SC-islets ( B ). ( C ) Heatmap of differentially expressed genes in VR versus control SC-islets. ( D, E ) Pathway analyses highlighting enrichment of apoptosis, cytokine signaling, and stress responses in SC-islets (PANTHER categories; box-and-whisker plots). ( F ) Confocal images of Annexin V staining in VR islets and SC-islets, indicating apoptosis. Scale bar = 75 µm. ( G ) qPCR analysis of apoptosis-related gene expression in VR-treated SC-islets (mean log₂ fold change [log₂FC], n = 3–4 per group; Student’s t -test, * = p < 0.05). Abbreviations: FDR, false discovery rate; HSR, heat shock response; ISR, integrated stress response; OSR, oxidative stress response; SC, stem cell; UPR, unfolded protein response; VR, vitrified and rewarmed.

Article Snippet: Then, 5× Annexin V binding buffer (Cat. #99902, Biotium) was diluted in deionized water to prepare 1× binding buffer.

Techniques: RNA Sequencing, Control, Whisker Assay, Staining, Gene Expression

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: BCoV infection induces the expression of PANoptosis-related genes in MDBK cells. A Western blot analysis of RIPK3, RIPK1, p-MLKL, and ZBP1 protein levels. B , C Relative mRNA levels of ZBP1 ( B ) and RIPK3 ( C ) determined by qPCR. D , E Protein ( D ) and mRNA ( E ) expression of CASP8. F Flow cytometric analysis of apoptosis via Annexin V-PE/7-AAD staining in control and BCoV-infected cells. G , H Protein ( G ) and mRNA ( H ) expression of GSDMD, CASP1, and ASC. I Relative mRNA expression of IL-1β . J , K Protein ( J ) and mRNA ( K ) expression of Neu1. L Cellular sialic acid levels, which decreased in a dose-dependent manner upon BCoV infection. Quantitative densitometry of the western blot results (normalized to β-actin/GAPDH) is shown in Additional file . The data are presented as the means ± SDs ( n = 3). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Expressing, Western Blot, Staining, Control

Journal: Veterinary Research

Article Title: Neu1 inhibition restrains BCoV replication and modulates ZBP1-dependent PANoptosis

doi: 10.1186/s13567-026-01729-7

Figure Lengend Snippet: The interaction between Neu1 and ZBP1 regulates apoptosis and sialic acid levels in BCoV-infected MDBK cells. A , B Schematic representation and validation of stable MDBK Neu1kd and MDBK ZBP1kd cell lines. C Apoptosis analysis of WT, MDBK Neu1kd , and MDBK ZBP1kd cells following BCoV infection via Annexin V-PE/7-AAD flow cytometry. D , E Viral titers determined via the Reed–Muench method ( D ) and relative BCoV mRNA expression levels ( E ). F Immunofluorescence localization of Neu1 (red) and ZBP1 (green) in WT, MDBK Neu1kd , and MDBK ZBP1kd cells. Nuclei were stained with DAPI (blue), and merged signals (yellow) indicate colocalization. G Cellular sialic acid levels significantly decreased in both MDBK Neu1kd and MDBK ZBP1kd cells. H Immunofluorescence analysis further demonstrated that Neu1 or ZBP1 knockdown markedly reduced the fluorescence signal intensity during BCoV infection. The data are presented as the means ± SDs ( n = 3). The quantification of fluorescence intensity is shown in Additional file . Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The pellets were washed three times with 1× PBS and subsequently resuspended in 1× Annexin V-PE binding buffer (0.1 M HEPES, 1.4 M NaCl, and 25 mM CaCl 2 solution; Servicebio, Wuhan) at the appropriate cell density.

Techniques: Infection, Biomarker Discovery, Flow Cytometry, Endpoint Dilution Assay, Expressing, Immunofluorescence, Staining, Knockdown, Fluorescence