dgat2 inhibitors t863  (Tocris)

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