Journal: Frontiers in Genetics

Article Title: Lyar contributes to cell cycle progression and multi-lineage differentiation in mouse embryonic stem cells

doi: 10.3389/fgene.2026.1786528

Figure Lengend Snippet: Establishing mESCs with functional loss of Lyar . (A) Schematic of Lyar gene targeting design. Depicted are the partial Lyar gene (Chr 5: NC_000071.7 , Exon 3–4) and its targeting vector, which carries left/right homologous arms (flanking Exon 3-4, matching PCR primer F/R sites), plus a PGK-driven Puro-P2A-mCherry cassette for Lyar locus modification. Primers F (Exon 3 upstream) and R (Exon 4 downstream) amplify the Exon 3-4 fragment. (B) PCR genotyping of Lyar locus in WT and clones 1–7: The 2165bp band indicates knockout (KO) allele, and 503bp band indicates WT allele. M, DNA ladder marker. (C) Western blot analysis showing expression levels of LYAR in WT and Lyar -KO mESCs. Full uncropped blots are provided in the . (D) Immunostaining of LYAR for WT and Lyar -KO mESCs. Scale bar, 50 μm.

Article Snippet: The primary antibodies used include anti-OCT4 (Proteintech, catalog no. 11263-1-AP), anti-NANOG (Proteintech, catalog no. 14295-1-AP) and LYAR Rabbit Polyclonal antibody (Proteintech, 24433-1-AP).

Techniques: Functional Assay, Plasmid Preparation, Modification, Clone Assay, Knock-Out, Marker, Western Blot, Expressing, Immunostaining

Journal: Frontiers in Genetics

Article Title: Lyar contributes to cell cycle progression and multi-lineage differentiation in mouse embryonic stem cells

doi: 10.3389/fgene.2026.1786528

Figure Lengend Snippet: Effects of Lyar knockout on the genomic stability. (A) Karyotype analysis of Lyar -KO mESCs after 12 passages. The Lyar KO clone exhibits a normal diploid karyotype (40 chromosomes, XY), consistent with WT mESC genetic background. (B) Morphological and fluorescence analysis of Lyar -KO mESCs. Bright-field images show that Lyar -KO mESCs maintain typical mESC colony morphology identical to WT cells. Fluorescence images reveal robust mCherry expression in Lyar -KO cells (but not in WT cells), confirming successful integration of the PGK-Puro-P2A-mCherry cassette. Scale bar, 100 μm.

Article Snippet: The primary antibodies used include anti-OCT4 (Proteintech, catalog no. 11263-1-AP), anti-NANOG (Proteintech, catalog no. 14295-1-AP) and LYAR Rabbit Polyclonal antibody (Proteintech, 24433-1-AP).

Techniques: Knock-Out, Fluorescence, Expressing

Journal: Frontiers in Genetics

Article Title: Lyar contributes to cell cycle progression and multi-lineage differentiation in mouse embryonic stem cells

doi: 10.3389/fgene.2026.1786528

Figure Lengend Snippet: Lyar is required for efficient proliferation of mESCs. Immunofluorescence staining of pluripotent marker NANOG (A) and OCT4 (B) in WT and Lyar -KO mESCs (left panels), and quantification of their mean fluorescence intensity (right panels). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. Data are presented as mean fluorescence intensity (arbitrary units, a.u.) ± SEM. Each dot represents an individual mESC colony. ns, not significant (Student’s t-test). (C) CCK-8 assay showing the proliferation rates of WT and Lyar- KO clone 1. (D) Cell cycle analysis. Left, representative flow cytometry dot plots showing EdU incorporation in WT and Lyar- KO clone 1 and clone 2; Right, quantification of EdU-positive cells. (E) Apoptosis analysis by Annexin V-APC/7-AAD staining. Left, representative flow cytometry plots of WT, Lyar -KO clone 1, and Lyar -KO clone 2. Right, quantification of the apoptotic rate. (F) Western blot analysis of key G1/S checkpoint regulators, including p53, p21, Cyclin D1, and p16, in WT, Lyar -KO clone 1, and Lyar -KO clone 2. Representative blots are shown. Full uncropped blots are provided in the . Data are presented as mean ± SEM (n = 3) and the significance level was calculated by Student’s t test (two-tailed, equal variance) (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: The primary antibodies used include anti-OCT4 (Proteintech, catalog no. 11263-1-AP), anti-NANOG (Proteintech, catalog no. 14295-1-AP) and LYAR Rabbit Polyclonal antibody (Proteintech, 24433-1-AP).

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, CCK-8 Assay, Cell Cycle Assay, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Frontiers in Genetics

Article Title: Lyar contributes to cell cycle progression and multi-lineage differentiation in mouse embryonic stem cells

doi: 10.3389/fgene.2026.1786528

Figure Lengend Snippet: Loss of Lyar alters the expression of lineage-specific markers during embryoid body differentiation of mESCs. (A) Morphology and mCherry expression of EBs derived from Lyar- KO and WT mESCs. Bright-field images show typical spherical EB morphology in both Lyar- KO and WT groups. Fluorescence images reveal stable mCherry expression in Lyar -KO EBs, while no fluorescence is detected in WT EBs (n = 3). Scale bar, 250 μm. (B) qPCR analysis of lineage-specific differentiation markers in EBs derived from WT and Lyar -KO clone 1 and clone 2 at day 0 (D0) and day 6 (D6) of differentiation. Markers for endoderm ( Gata4 , Sox17 ), mesoderm ( Gsc , T ), and ectoderm ( Pax6 , Nestin ) were examined. For each time point, the expression levels of Lyar -KO groups were individually normalized to those of the WT group (D0 Lyar -KO vs. D0 WT; D6 Lyar -KO vs. D6 WT). Data are presented as mean ± SEM (n = 3) and the significance level was calculated by Student’s t test (two-tailed, equal variance) (ns, not statistically significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Article Snippet: The primary antibodies used include anti-OCT4 (Proteintech, catalog no. 11263-1-AP), anti-NANOG (Proteintech, catalog no. 14295-1-AP) and LYAR Rabbit Polyclonal antibody (Proteintech, 24433-1-AP).

Techniques: Expressing, Derivative Assay, Fluorescence, Two Tailed Test