Fig 1: Processing body disassembly is not a common feature of all human coronavirus N proteins.A-D. HUVECs were transduced with recombinant lentiviruses ectopically expressing N protein from the Betacoronaviruses MERS-CoV and OC43 (A-B) or N protein from Alphacoronaviruses 229E and NL63 (C-D) or N protein from SARS-CoV-1 (E-F). A control lentiviral expressing an empty vector (EV) was used as a negative control and SARS-CoV-2 N protein expressing lentiviruses were used as a positive control in each experiment. Cells were selected, fixed and immunostained for DDX6 (PBs; white; Alexa555) and either authentic N protein or a FLAG tag (green; Alexa488). Nuclei were stained with Hoechst (blue). Scale bar = 20 μm. DDX6 puncta in EV or N-transduced cells were quantified using CellProfiler as in Fig 1. DDX6 puncta were quantified as in Fig 1. Representative images from one independent experiment of three are shown. These data represent three independent biological replicates (n = 3) with >30 cells measured per condition (EV and N) per replicate. Each EV and N replicate pair plotted independently; mean. Statistics were performed using Kruskal-Wallis H test with Dunn’s correction (*, p < 0.0332; **, p < 0.0021; ****, p < 0.0001; ns, nonsignificant). G-I. HUVECs were transduced as above, protein lysate was harvested and immunoblotting was performed using XRN1, Hedls, DCP1A, DDX6, N protein or FLAG, and β-actin specific antibodies. One representative experiment of three is shown.
Fig 2: Coronavirus infection does not alter steady state levels of most processing body proteins.A. HUVECs were transduced with human ACE2 (HUVECACE2), selected, and infected with SARS-CoV-2 TO-1 isolate (MOI = 3). Cells were lysed at 6 and 12 hours post infection and immunoblotting was performed using XRN1, Hedls, DCP1A, DDX6, SARS-CoV-2 N, and β-actin specific antibodies. One representative experiment of two is shown. B-C. HUVECs were infected with OC43 (B, TCID50 = 2 x 104) or 229E (C, TCID50 = 2.4 x 103). Cells were lysed at 12 and 24 hours post infection (B, OC43) or 6 and 12 hours post infection (C, 229E). Immunoblotting was performed using XRN1, Hedls, DCP1A, DDX6, OC43 N protein (B only), and β-actin specific antibodies. One representative experiment of three is shown.
Fig 3: Dcp1a protein levels are decreased by KapB expression and Torin treatment.A: HUVECs were treated with DMSO or Torin (250 nM) for 4 h prior to harvest. Samples were lysed in 2X Laemmli buffer and resolved by SDS-PAGE before immunoblotting for Xrn1, EDC4/Hedls, Dcp1a, and DDX6. Samples were quantified by normalizing the PB resident protein levels to the total protein in each lane and then the DMSO control using ImageLab (BioRad). Results were plotted in GraphPad and a one-way ANOVA was performed ±SEM; n = 3, ** = P<0.01. B: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Cells were treated with DMSO or Bafilomycin A1 (BafA1, 10 nM) for 4 h prior to harvest in 2X Laemmli buffer. Samples were resolved by SDS-PAGE and immunoblot was performed for Dcp1a or p62 (autophagy marker). Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector DMSO control. Representative blot is from the same membrane, hashed line indicates skipped lanes. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, *** = P<0.001. C: HUVECs were transduced with recombinant lentiviruses expressing either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer, resolved by SDS-PAGE and immunoblot was performed for Xrn1, Hedls/EDC4, or DDX6. Samples were quantified by normalizing PB protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector control. Results were plotted in GraphPad. D: HUVECs were sequentially transduced: first with recombinant lentiviruses expressing either shRNAs targeting Atg5 (shAtg5) or a non-targeting control (NS) and selected with puromycin (1 μg/mL), and second with either KapB or an empty vector control and selected with blasticidin (5 μg/mL). Samples were harvested in 2X Laemmli buffer and resolved by SDS-PAGE. Immunoblot was performed for Dcp1a, Atg5, and KapB. Samples were quantified by normalizing Dcp1a protein levels to the total protein in each lane using Image Lab (BioRad) and then to the vector NS control. Results were plotted in GraphPad and a 2-way ANOVA was performed, ±SEM; n = 3, * = P<0.05, **P = <0.01.
Supplier Page from Abcam for Anti-Xrn1 antibody