Journal: Journal of Animal Science and Biotechnology

Article Title: METTL3 regulates Leydig cell proliferation via miR-145-PCK1 mediated gluconeogenesis in goats

doi: 10.1186/s40104-025-01307-5

Figure Lengend Snippet: METTL3 regulates goat LC proliferation through the glyconeogenesis pathway. A Cell viability of LCs treated with siMETTL3 and siMETTL3+3-MPA group ( n = 5). B LCs were treated with siMETTL3 and siMETTL3+3-MPA, and EdU (green) was used to label the proliferating cells. The cell nuclei were counterstained with DAPI ( n = 4). Scale bar: 100 μm. C Lactic acid level of LCs treated with siNC and siMETTL3+3-MPA for 24 h ( n = 4). D Glucose level of LCs treated with siMETTL3 and siMETTL3+3-MPA for 24 h ( n = 4). E Glucose uptake ability of LCs treated with siMETTL3 and siMETTL3+3-MPA, assessed using immunofluorescence and quantitative analysis ( n = 4). Scale bar: 100 μm. F – G Changes in mitochondrial membrane potential in LCs treated with siMETTL3 and siMETTL3+3-MPA were analyzed by JC-1 staining and the fluorescence intensity of JC-1 aggregates/monomers was quantified ( n = 4). Scale bar: 100 μm. H The relative mRNA expression levels of METTL3 , PCK1 , CCNB1 and CCNE2 treated with siMETTL3 and siMETTL3+3-MPA ( n = 3). I – J The levels of METTL3, PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). * P < 0.05, ** P < 0.01

Article Snippet: To determine whether METTL3 induces proliferation through the gluconeogenic pathway, a 10 μmol/L dose of the gluconeogenic inhibitor 3-MPA (MCE, UK) was added 12 h after transfection along with siMETTL3, forming the siMETTL3 + 3-MPA group.

Techniques: Immunofluorescence, Membrane, Staining, Fluorescence, Expressing, Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: METTL3 regulates Leydig cell proliferation via miR-145-PCK1 mediated gluconeogenesis in goats

doi: 10.1186/s40104-025-01307-5

Figure Lengend Snippet: miR-145-3p regulates goat LC proliferation through the glyconeogenesis pathway. A Cell viability of LCs treated with Mimics-NC and Mimics-145-3p ( n = 5). B LCs were treated with Mimics-NC and Mimics-145-3p, and EdU (green) was used to label the proliferating cells. The cell nuclei were counterstained with DAPI ( n = 4). Scale bar: 100 μm. C Glucose level of LCs treated with Mimics-NC and Mimics-145-3p ( n = 4). D Lactic acid level of LCs treated with Mimics-NC and Mimics-145-3p ( n = 4). E – F Glucose uptake ability of LCs treated with Mimics-NC and Mimics-145-3p, assessed using immunofluorescence and quantitative analysis ( n = 4). Scale bar: 100 μm. G – H Changes in mitochondrial membrane potential in LCs treated with Mimics-NC and Mimics-145-3p were analyzed by JC-1 staining and the fluorescence intensity of JC-1 aggregates/monomers was quantified ( n = 4). Scale bar: 100 μm. I The relative mRNA expression levels of PCK1 , CCNB1 and CCNE2 treated with Mimics-NC and Mimics-145-3p ( n = 3). J – K The levels of PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). L Cell viability of LCs treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA ( n = 5). M LCs were treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA, and EdU (green) was used to label the proliferating cells. The cell nuclei were counterstained with DAPI ( n = 4). Scale bar: 100 μm. N Glucose level of LCs treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA ( n = 4). O Lactic acid level of LCs treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA ( n = 4). P – Q Glucose uptake ability of LCs treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA, assessed using immunofluorescence and quantitative analysis ( n = 4). Scale bar: 100 μm. R – S Changes in mitochondrial membrane potential in LCs treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA were analyzed by JC-1 staining and the fluorescence intensity of JC-1 aggregates/monomers was quantified ( n = 4). Scale bar: 100 μm. T The relative mRNA expression levels of CCNB1 and CCNE2 treated with Inhibitor-145-3p and Inhibitor-145-3p+3-MPA ( n = 3). U – V The levels of PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). * P < 0.05, ** P < 0.01

Article Snippet: To determine whether METTL3 induces proliferation through the gluconeogenic pathway, a 10 μmol/L dose of the gluconeogenic inhibitor 3-MPA (MCE, UK) was added 12 h after transfection along with siMETTL3, forming the siMETTL3 + 3-MPA group.

Techniques: Immunofluorescence, Membrane, Staining, Fluorescence, Expressing, Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: METTL3 regulates Leydig cell proliferation via miR-145-PCK1 mediated gluconeogenesis in goats

doi: 10.1186/s40104-025-01307-5

Figure Lengend Snippet: PCK1 regulates goat Leydig cell proliferation through the gluconeogenesis pathway. A The relative mRNA expression levels of PCK1 , CCNB1 and CCNE2 treated with OE-NC and OE-PCK1 ( n = 3). B – C The levels of PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). D The relative mRNA expression levels of PCK1 , CCNB1 and CCNE2 treated with siNC and siPCK1 ( n = 3). E – F The levels of PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). G Cell viability of LCs treated with OE-PCK1 and OE-PCK1+3-MPA group ( n = 5). H LCs were treated with OE-PCK1 and OE-PCK1+3-MPA group, and EdU (green) was used to label the proliferating cells. The cell nuclei were counterstained with DAPI ( n = 4). Scale bar: 100 μm. I Glucose level of LCs treated with OE-PCK1 and OE-PCK1+3-MPA ( n = 4). J Lactic acid of LCs treated with OE-PCK1 and OE-PCK1+3-MPA ( n = 4). K–L Glucose uptake ability of LCs treated with OE-PCK1 and OE-PCK1+3-MPA, assessed using immunofluorescence and quantitative analysis ( n = 4). Scale bar: 100 μm. M–N Changes in mitochondrial membrane potential in LCs treated with OE-PCK1 and OE-PCK1+3-MPA were analyzed by JC-1 staining and the fluorescence intensity of JC-1 aggregates/monomers was quantified ( n = 4). Scale bar: 100 μm. O The relative mRNA expression levels of CCNB1 and CCNE2 treated with OE-PCK1 and OE-PCK1+3-MPA ( n = 3). P – Q The levels of PCK1, CCNB1, CCNE2 and β-actin were assessed via Western blotting. Relative protein levels were analysed via gray scanning ( n = 3). * P < 0.05, ** P < 0.01

Article Snippet: To determine whether METTL3 induces proliferation through the gluconeogenic pathway, a 10 μmol/L dose of the gluconeogenic inhibitor 3-MPA (MCE, UK) was added 12 h after transfection along with siMETTL3, forming the siMETTL3 + 3-MPA group.

Techniques: Expressing, Western Blot, Immunofluorescence, Membrane, Staining, Fluorescence

Journal: ACS Omega

Article Title: Infectious cDNA Clone of Chikungunya Virus 181/25 for Antiviral Assay Development

doi: 10.1021/acsomega.5c09397

Figure Lengend Snippet: Validation of the viral infectivity inhibition assay using known chikungunya inhibitors. (A) Dose response curves and % cell viability of mycophenolic acid (MPA), Arbidol, epigallocatechin gallate (EGCG), Suramin, and 6-Azauridine. Vero cells were infected with 1MOI of GFP-wtCHIKV in a 96-well plate pretreated with compounds as detailed in the section. The cells were harvested and processed for FACS, and % GFP-positive cells were measured in each well. The values are plotted as % viral infection against different drug concentrations (50–0.78 μM) (Blue line). MTT assay was run on Vero cells, and values were plotted against different drug concentrations (100–1.56 μM) (red line). The values are presented as mean with SD. The values are from two different experiments run with triplicate technical repeats. The experiment was run more than three times with similar results. (B) Table containing IC50 and CC50 values and 95% confidence intervals calculated using nonlinear regression analysis in GraphPad Prism (8.0). ND: nondetected at the tested concentration. (C) Immunofluorescence analysis of inhibition in GFP-wtCHIKV infectivity in the presence of different concentrations of MPA. The BHK21 cells were treated with MPA and infected with GFP-wtCHIKV as mentioned above, and pictures were taken 24 h PI. Representative images are shown from more than three technical repeats. The images were taken at 10× objective magnification with a fluorescence microscope (Nikon).

Article Snippet: The compounds mycophenolic acid (MPA), suramin, 6-azauridine, EGCG, and Arbidol used to validate the antiviral assay were procured from MedChemExpress (NJ, USA).

Techniques: Biomarker Discovery, Infection, Inhibition, MTT Assay, Concentration Assay, Immunofluorescence, Fluorescence, Microscopy