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t863  (MedChemExpress)


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    MedChemExpress t863
    LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of <t>T863</t> and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    T863, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 93/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 19 article reviews
    t863 - by Bioz Stars, 2026-05
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    Images

    1) Product Images from "African swine fever virus hijacks lipolysis induced by chaperone-mediated autophagy to upregulate fatty acid β-oxidation and promote viral replication"

    Article Title: African swine fever virus hijacks lipolysis induced by chaperone-mediated autophagy to upregulate fatty acid β-oxidation and promote viral replication

    Journal: mBio

    doi: 10.1128/mbio.03368-25

    LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of T863 and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
    Figure Legend Snippet: LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of T863 and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Techniques Used: Solvent, Control, Staining, Fluorescence, Microscopy, Software, Western Blot, Infection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Negative Control



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    LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of <t>T863</t> and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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    LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of <t>T863</t> and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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    The EGFP-tagged 3a protein does not affect lipid droplet (LD) accumulation, but nsp6 enhances it; both mediate cell size reduction. ( A ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in a cell culture transfected with pDNA encoding the viral EGFP-tagged 3a protein (3a-EGFP, green) either in a cell without apparent 3a expression (CTL 3a-EGFP; n =9; 116 cells) or in a cell expressing 3a (3a-EGFP; n =9; 151 cells) and labelled with fluorescent LD marker HCS LipidTOX red (red) and nuclear marker 4′,6-diamidino-2-phenylindole (DAPI, blue) 24 h after transfection. The overlay presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars, 20 µm and 5 µm (inset). ( B ) Histograms of mean LipidTOX-stained cell area normalized to untreated controls (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( C ) mean cell area and mean cell perimeter in CTL, CTL 3a-EGFP and 3a-EGFP astrocytes. Data are presented as means±SEM; n , number of independent experiments; *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [cell perimeter]). ( D ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( E ) mean cell area and mean cell perimeter of cortical astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in non-transfected cell cultures on 24-h exposure to 10 µM ketamine (KM; n =7; 84 cells), in cell cultures transfected with pDNA encoding the viral 3a protein (3a-EGFP; n =9; 151 cells) and in cell cultures transfected with pDNA encoding 3a on 24 h exposure to 10 µM KM (3a-EGFP+KM; n =8; 134 cells). Data are presented as means±SEM; n , number of independent experiments. * P <0.05, ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [LD area, LD diameter, cell perimeter]). ( F ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 111 cells), in a cell culture transfected with pDNA encoding viral nsp6 protein, either in a cell without apparent nsp6 expression (CTL nsp6; n =15; 228 cells) or in a cell expressing nsp6 (nsp6; n =15; 244 cells), and nsp6-expressing astrocytes in a cell culture treated with DGAT1 and <t>DGAT2</t> inhibitors (nsp6+DGATi; 10 µM; n =5; 56 cells). To trace nsp6 expression, LD formation and the presence of nuclei, cell cultures were labelled immunocytochemically with antibodies against nsp6 (Alexa Fluor 488, green), fluorescent LD marker HCS LipidTOX red (red) and nuclear marker DAPI (blue) 24 h on transfection. Overlays presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars: 20 µm and 5 µm (inset). ( G ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( H ) mean cell area and mean cell perimeter in CTL, CTL nsp6, nsp6 and nsp6+DGATi astrocytes. Data are presented as means±SEM. n , number of independent experiments. ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [LD diameter, cell area, cell perimeter] and ANOVA on ranks, Dunn’s test [LD area]). ( I ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( J ) mean cell area and mean cell perimeter of astrocytes in non-transfected cell cultures without treatment (CTL; n =3, 51 cells) and on 24-h treatment with 10 µM KM ( n =4; 50 cells), and of nsp6-expressing astrocytes without treatment (nsp6; n =5; 62 cells) and on 24-h treatment with 10 µM KM (nsp6+KM; n =4; 52 cells). Data are presented as means±SEM; n , number of independent experiments; * P <0.05, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test).
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    LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of T863 and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: mBio

    Article Title: African swine fever virus hijacks lipolysis induced by chaperone-mediated autophagy to upregulate fatty acid β-oxidation and promote viral replication

    doi: 10.1128/mbio.03368-25

    Figure Lengend Snippet: LDs facilitate ASFV replication. ( A and B ) OA induced LD biogenesis in PAMs. PAMs were treated with OA (80 µM) for 24 h, and the same volume of the solvent was added as a control. LDs were stained with Bodipy 493/503 (green), nucleus was stained with DAPI (blue), and the images were captured by a fluorescence microscope. The fluorescence intensity of LDs was quantified using Image J software ( A ). The protein levels of PLIN2 and β-actin were analyzed by western blotting ( B ). ( C and D ) OA promotes ASFV replication. PAMs were treated with increasing concentrations of OA (0, 20, 40, and 80 µM) for 24 h, followed by infection with ASFV (MOI = 0.1) for an additional 24 h. Western blotting was then performed to determine the expression levels of p30, p72, and β-actin ( C ), while RT-qPCR was employed to analyze p72 mRNA levels ( D ). ( E ) OA enhanced viral titers of ASFV in PAMs. PAMs were treated with OA (80 µM) for 24 h and infected with ASFV (MOI = 0.1) for another 24 h. The viral titers were determined by the HAD assay. ( F and G ) The effect of T863 and PF-06424439 on cell viability. PAMs were treated with different concentrations of PF-06424439 (12.5, 25, 50, and 100 µM) and T863 (12.5, 25, 50 and 100 µM) for 24 h, and cell viability was analyzed through the CCK-8 assay. (H–J) T863 and PF-06424439 inhibited ASFV replication on PAMs. PAMs were treated with PF-06424439 (50 µM) and/or T863 (50 µM) and infected with ASFV (MOI = 0.1) for 24 h. The same volume of DMSO was added as a negative control. Viral replication was determined by western blotting ( H ) and RT-qPCR ( I ). The viral titers of PF-06424439 (50 µM) and T863 (50 µM) combined were determined by the HAD assay ( J ). ( K ) Effect of OA on ASFV-GFP replication in PAMs. PAMs were treated with OA (80 µM) or the same volume of the solvent for 24 h, followed infection with ASFV-GFP (MOI = 0.1). Images were captured under a fluorescence microscope, and the GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. ( L ) Effect of DGAT inhibitors on ASFV replication in PAMs. PAMs were treated with PF-06424439 (50 µM) and T863 (50 µM) for 24 h, and DMSO was used as a blank control. The samples were mock-infected and ASFV-infected (MOI = 0.1) for another 24 h, and the images were captured under a fluorescence microscope. The GFP fluorescence intensity was quantified using Image J software. Scale bars = 300 µm. Data are presented as the means ± SDs of three independent experiments and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: NH 4 Cl (HY-Y1269), PF-06424439 (HY-108341), T863 (HY-32219), BODIPY 493/503 (HY-W090090), and BODIPY 558/568 C12 (HY-138226) were purchased from MedChemExpress.

    Techniques: Solvent, Control, Staining, Fluorescence, Microscopy, Software, Western Blot, Infection, Expressing, Quantitative RT-PCR, CCK-8 Assay, Negative Control

    The EGFP-tagged 3a protein does not affect lipid droplet (LD) accumulation, but nsp6 enhances it; both mediate cell size reduction. ( A ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in a cell culture transfected with pDNA encoding the viral EGFP-tagged 3a protein (3a-EGFP, green) either in a cell without apparent 3a expression (CTL 3a-EGFP; n =9; 116 cells) or in a cell expressing 3a (3a-EGFP; n =9; 151 cells) and labelled with fluorescent LD marker HCS LipidTOX red (red) and nuclear marker 4′,6-diamidino-2-phenylindole (DAPI, blue) 24 h after transfection. The overlay presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars, 20 µm and 5 µm (inset). ( B ) Histograms of mean LipidTOX-stained cell area normalized to untreated controls (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( C ) mean cell area and mean cell perimeter in CTL, CTL 3a-EGFP and 3a-EGFP astrocytes. Data are presented as means±SEM; n , number of independent experiments; *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [cell perimeter]). ( D ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( E ) mean cell area and mean cell perimeter of cortical astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in non-transfected cell cultures on 24-h exposure to 10 µM ketamine (KM; n =7; 84 cells), in cell cultures transfected with pDNA encoding the viral 3a protein (3a-EGFP; n =9; 151 cells) and in cell cultures transfected with pDNA encoding 3a on 24 h exposure to 10 µM KM (3a-EGFP+KM; n =8; 134 cells). Data are presented as means±SEM; n , number of independent experiments. * P <0.05, ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [LD area, LD diameter, cell perimeter]). ( F ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 111 cells), in a cell culture transfected with pDNA encoding viral nsp6 protein, either in a cell without apparent nsp6 expression (CTL nsp6; n =15; 228 cells) or in a cell expressing nsp6 (nsp6; n =15; 244 cells), and nsp6-expressing astrocytes in a cell culture treated with DGAT1 and DGAT2 inhibitors (nsp6+DGATi; 10 µM; n =5; 56 cells). To trace nsp6 expression, LD formation and the presence of nuclei, cell cultures were labelled immunocytochemically with antibodies against nsp6 (Alexa Fluor 488, green), fluorescent LD marker HCS LipidTOX red (red) and nuclear marker DAPI (blue) 24 h on transfection. Overlays presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars: 20 µm and 5 µm (inset). ( G ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( H ) mean cell area and mean cell perimeter in CTL, CTL nsp6, nsp6 and nsp6+DGATi astrocytes. Data are presented as means±SEM. n , number of independent experiments. ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [LD diameter, cell area, cell perimeter] and ANOVA on ranks, Dunn’s test [LD area]). ( I ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( J ) mean cell area and mean cell perimeter of astrocytes in non-transfected cell cultures without treatment (CTL; n =3, 51 cells) and on 24-h treatment with 10 µM KM ( n =4; 50 cells), and of nsp6-expressing astrocytes without treatment (nsp6; n =5; 62 cells) and on 24-h treatment with 10 µM KM (nsp6+KM; n =4; 52 cells). Data are presented as means±SEM; n , number of independent experiments; * P <0.05, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test).

    Journal: bioRxiv

    Article Title: Ketamine inhibition of SARS-CoV-2 replication in astrocytes is associated with COVID-19 disease severity in a variant-dependent manner

    doi: 10.1101/2025.03.29.646083

    Figure Lengend Snippet: The EGFP-tagged 3a protein does not affect lipid droplet (LD) accumulation, but nsp6 enhances it; both mediate cell size reduction. ( A ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in a cell culture transfected with pDNA encoding the viral EGFP-tagged 3a protein (3a-EGFP, green) either in a cell without apparent 3a expression (CTL 3a-EGFP; n =9; 116 cells) or in a cell expressing 3a (3a-EGFP; n =9; 151 cells) and labelled with fluorescent LD marker HCS LipidTOX red (red) and nuclear marker 4′,6-diamidino-2-phenylindole (DAPI, blue) 24 h after transfection. The overlay presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars, 20 µm and 5 µm (inset). ( B ) Histograms of mean LipidTOX-stained cell area normalized to untreated controls (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( C ) mean cell area and mean cell perimeter in CTL, CTL 3a-EGFP and 3a-EGFP astrocytes. Data are presented as means±SEM; n , number of independent experiments; *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [cell perimeter]). ( D ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( E ) mean cell area and mean cell perimeter of cortical astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 84 cells), in non-transfected cell cultures on 24-h exposure to 10 µM ketamine (KM; n =7; 84 cells), in cell cultures transfected with pDNA encoding the viral 3a protein (3a-EGFP; n =9; 151 cells) and in cell cultures transfected with pDNA encoding 3a on 24 h exposure to 10 µM KM (3a-EGFP+KM; n =8; 134 cells). Data are presented as means±SEM; n , number of independent experiments. * P <0.05, ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [cell area] and ANOVA on ranks, Dunn’s test [LD area, LD diameter, cell perimeter]). ( F ) Representative fluorescence images of astrocytes in non-transfected and untreated cell cultures (CTL; n =7; 111 cells), in a cell culture transfected with pDNA encoding viral nsp6 protein, either in a cell without apparent nsp6 expression (CTL nsp6; n =15; 228 cells) or in a cell expressing nsp6 (nsp6; n =15; 244 cells), and nsp6-expressing astrocytes in a cell culture treated with DGAT1 and DGAT2 inhibitors (nsp6+DGATi; 10 µM; n =5; 56 cells). To trace nsp6 expression, LD formation and the presence of nuclei, cell cultures were labelled immunocytochemically with antibodies against nsp6 (Alexa Fluor 488, green), fluorescent LD marker HCS LipidTOX red (red) and nuclear marker DAPI (blue) 24 h on transfection. Overlays presents merged green, red and blue channels. DIC presents images taken by differential-interference contrast microscopy. The white line in the images shows the outline of an individual cell. Insets show white-boxed regions at higher magnification. Scale bars: 20 µm and 5 µm (inset). ( G ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( H ) mean cell area and mean cell perimeter in CTL, CTL nsp6, nsp6 and nsp6+DGATi astrocytes. Data are presented as means±SEM. n , number of independent experiments. ** P <0.01, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test [LD diameter, cell area, cell perimeter] and ANOVA on ranks, Dunn’s test [LD area]). ( I ) Histograms of mean LipidTOX-stained cell area normalized to control untreated samples (LD area [fold change]), mean diameter of LipidTOX-stained LDs (LD diameter), and ( J ) mean cell area and mean cell perimeter of astrocytes in non-transfected cell cultures without treatment (CTL; n =3, 51 cells) and on 24-h treatment with 10 µM KM ( n =4; 50 cells), and of nsp6-expressing astrocytes without treatment (nsp6; n =5; 62 cells) and on 24-h treatment with 10 µM KM (nsp6+KM; n =4; 52 cells). Data are presented as means±SEM; n , number of independent experiments; * P <0.05, *** P <0.001 all pairwise (ANOVA, Holm-Sidak test).

    Article Snippet: After transfection, astrocytes were incubated at 37°C for 24 h or 48 h. In some experiments, on transfection, astrocytes were supplied with growth medium containing DGAT1 and DGAT2 inhibitors T863 (10 µM) and PF-06424439 (10 µM), respectively, to prevent biogenesis of LD, or with growth medium containing ketamine (10 µM; Tocris Bioscience, Bristol, UK) and incubated at 37°C for 24 h.

    Techniques: Fluorescence, Transfection, Cell Culture, Expressing, Marker, Microscopy, Staining, Control