Journal: bioRxiv

Article Title: Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

doi: 10.1101/2023.11.07.566110

Figure Lengend Snippet: a , Representative confocal images show three types of vgRNA distribution in SARS-CoV-2 infected cells. b , Number of cells assigned to one of the three types at 6 or 24 hpi. c , Cell-integrated vgRNA signal increases significantly from 6 hpi to 24 hpi. p-value = 6⋅10 -8 , two-tailed t-test. d - e , Cell-integrated signal of immunofluorescently detected dsRNA ( d ) and nsp12 ( e ) in SARS-CoV-2 infected cells does not significantly change from 6 hpi to 24 hpi. p-values = 0.13 ( d ) and 0.23 ( e ), two-tailed t-test. f , dsRNA signal correlates with vgRNA signal at 6 hpi (Pearson’s r = 0.76). g , dsRNA signal does not correlate with vgRNA signal at 24 hpi (Pearson’s r = 0.18). Error bars represent mean + SD of the values from individual cells. Scale bars, 10 µm.

Article Snippet: Primary antibodies and the optimal dilutions and concentrations used are as follows: goat polyclonal anti-spike S2 (Novus Biologicals, AF10774-SP, 1:20, 10 µg/mL), mouse monoclonal anti-dsRNA (SCICONS, 10010200, 1:200, 5 µg/mL), rabbit polyclonal anti-RdRp/nsp12 (Sigma-Aldrich, SAB3501287-100UG, 1:500, 2 µg/mL), mouse monoclonal anti-nucleocapsid (Thermo Fisher, MA5-29981, 1:500, 2 µg/mL), rabbit polyclonal anti-nsp3 (Thermo Fisher, PA5-116947, 1:134, 5 µg/mL), sheep polyclonal anti-GFP (Bio-Rad, 4745-1051, 1:1000, 5 µg/mL), and rabbit polyclonal anti-GFP (Novus Biologicals, NB600-308SS, 1:163, 5 µg/mL).

Techniques: Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

doi: 10.1101/2023.11.07.566110

Figure Lengend Snippet: a - b , Representative confocal images of SARS-CoV-2 infected cells display DL colocalization between nsp12, the catalytic subunit of RdRp (green) and vgRNA (magenta) at both 6 hpi ( a ) and 24 hpi ( b ). c - d , Representative SR images of SARS-CoV-2 infected cells indicate nanoscale association between nsp12 and vgRNA at both 6 hpi ( c ) and 24 hpi ( d ). Insets show magnified images of corresponding regions in colored boxes. e - f , Bivariate pair-correlation functions peak at r = 0 nm indicating association between nsp12 and vgRNA. g , Number of nanoscale puncta of nsp12 per vgRNA cluster. h , SR image of nsp12 with Sec61β suggests encapsulation of nsp12 within ER-derived membranes. Scale bars, 10 µm ( a - b ), 1 µm ( c - d ), 500 nm ( h and insets in c - d ). Dashed lines in c , d and h indicate the edge of the cell nucleus.

Article Snippet: Primary antibodies and the optimal dilutions and concentrations used are as follows: goat polyclonal anti-spike S2 (Novus Biologicals, AF10774-SP, 1:20, 10 µg/mL), mouse monoclonal anti-dsRNA (SCICONS, 10010200, 1:200, 5 µg/mL), rabbit polyclonal anti-RdRp/nsp12 (Sigma-Aldrich, SAB3501287-100UG, 1:500, 2 µg/mL), mouse monoclonal anti-nucleocapsid (Thermo Fisher, MA5-29981, 1:500, 2 µg/mL), rabbit polyclonal anti-nsp3 (Thermo Fisher, PA5-116947, 1:134, 5 µg/mL), sheep polyclonal anti-GFP (Bio-Rad, 4745-1051, 1:1000, 5 µg/mL), and rabbit polyclonal anti-GFP (Novus Biologicals, NB600-308SS, 1:163, 5 µg/mL).

Techniques: Infection, Encapsulation, Derivative Assay