Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Serum antibody features in COVID-19 patients during hospitalization: Graphs show serum anti-S-RBD IgM ( A ) and IgG ( B ) levels for each disease severity ( n = 9 patients/mild group; 10 patients/moderate I group; 9 patients/moderate II patients). Blue and red dots indicate admission and discharge, respectively; the duration of hospitalization is indicated at the bottom of each panel. Graphs show the correlation between anti-S-RBD IgM or IgG and neutralizing antibody titer ( C ) and age, as well as the change in the titer against the WT strain in patients infected with the WT and VOCs ( D ). Each dot denotes disease severity, and Spearman’s correlation coefficients are shown within the graph. Gray areas indicate a 95% confidence interval (CI) for the total. ( E ) Dot plot indicates the neutralizing potency against the WT strain by sex. Graphs show the serum-neutralizing antibody titer against WT ( F ), Kappa ( G ), and Delta ( H ) variants at each severity (moderate II is classified by the infected strains, n = 9 patients/mild group; 10 patients/moderate I group; 9 patients/WT-infected moderate II group; 8 patients/VOC-infected moderate II group). Statistical significances were determined using the Tukey–Kramer test and Welch’s t -test. All horizontal bars show mean values. Cross, deceased; #, long-term observed healthcare worker. * p < 0.05; *** p < 0.005; ns, not significant.

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Infection

Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Longitudinal analyses of serum neutralizing antibody activities and PBMCs in a patient with high neutralizing potency: ( A ) Concentration of anti-S-RBD IgM and IgG in the serum of a healthcare worker (indicated by # in ) upon infection. ( B ) Fluctuation of each neutralizing antibody titer from onset to post-vaccination ( n = 1). ( C ) Representative FACS plots of cell population are shown in panels. The numbers indicate the positive rate of each cell population. Graphs show the absolute number of IgG + S-RBD + Bmems ( D ) and the frequency of lymphocyte ( E ), CD19 + CD27 + CD38 high , and IgG + Bmems (( F ); n = 1, 3–5 technical replicates). Black dots indicate technical replicates. Each number indicates the mean.

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Concentration Assay, Infection

Journal: Vaccines

Article Title: Longitudinal Analyses after COVID-19 Recovery or Prolonged Infection Reveal Unique Immunological Signatures after Repeated Vaccinations

doi: 10.3390/vaccines10111815

Figure Lengend Snippet: Affinity maturation of S-RBD-specific memory B-cells after vaccination: Pie charts show the distribution of heavy chain genes ( A ) and light chain genes ( B ) from onset to post-vaccination, comparing patients with the recovered individual ( n = 1) and a prolonged COVID-19 patient ( n = 1). The number in the inner circle indicates the number of sequenced clones. ( C ) Graphs show the number of somatic hypermutations in each antibody gene. Statistical significances were determined using Tukey–Kramer test. ( D ) Graphs show the distribution of CDR3 length in each antibody gene (heavy chains; n = 34–45 clones/recovered individual, 14 clones/prolonged COVID-19 patient; light chains; 18–66 clones/recovered individual, 15 clones/prolonged COVID-19 patient).

Article Snippet: Isolated PBMCs were incubated with His-tagged SARS-CoV-2 S-RBD recombinant protein (Cell Signaling Technology, Danvers, MA, USA) for 30 min on ice.

Techniques: Clone Assay

Journal: bioRxiv

Article Title: Cutaneous jet-injection of naked mRNA vaccine induces robust immune responses without systemic vaccine spillage

doi: 10.1101/2023.02.27.530188

Figure Lengend Snippet: (a-d) Balb/C mice were PYRO-injected with naked spike mRNA at both flanks in a prime-boost setting separated by a 3-week interval. 5 weeks after the prime, blood plasma and splenocytes were collected for evaluating humoral and cellular immunity. (a,b) Anti-spike IgG ELISA absorbance vs. plasma dilution curves . (c) The ability of plasma in blocking the binding between spike-RBD and ACE2 proteins. (d) Quantification of spike-specific INFγ-positive splenocytes. Data represent the mean ± SEM (n=4). **p <0.01, *p <0.05 vs. NT, non-repeated ANOVA followed by Dunnett’s test in (d) . (e-l) Vaccination in NHPs. (e) Injections and sampling schedule. (f,g) Appearance of PYRO injection site on the back of Cynomolgus monkeys (f) compared to Balb/C mice (g) . (h) Anti-spike IgG titers in NHPs PYRO-injected with buffer or mRNA solution. (i) Binding inhibition of ACE2 and SARS-CoV-2 RBD by plasma of immunized monkeys. (j-l) Toxicity evaluation in PYRO-injecting monkeys receiving either buffer or spike mRNA. (j) Body temperature measured immediately before and 24 h post dosing, after each of the 3 doses. (k) Change in body weight. (l) Plasma levels of proinflammatory cytokines before and 24 h after the first dose. n.s.: nonsignificant, **p <0.01, *p <0.05 vs. buffer treatment in unpaired Student’s t -test. BLQ: below limit of quantification.

Article Snippet: SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit was obtained from Cell Signaling Technologies.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Sampling, Inhibition

Journal: The Journal of Physical Chemistry. B

Article Title: Machine Learning Guided Design of High-Affinity ACE2 Decoys for SARS-CoV-2 Neutralization

doi: 10.1021/acs.jpcb.3c00469

Figure Lengend Snippet: Performance of ACE2 double mutants predicted by TLmutation. Expi293F cells expressing wild type or mutant myc-ACE2 were coincubated with a fluorescent anti-myc antibody and with RBD-sfGFP expression medium at a subsaturating dilution. After gating for a constant level of anti-myc fluorescence to control for expression differences, the bound RBD-sfGFP was measured by flow cytometry. Data are mean, N = 2, error bars represent range. The double mutants belonged to four groups and are colored accordingly: blue, mutants that scored in the top 1% of TLmutation; red, that scored in the lowest 1% of TLmutation; yellow, double mutants whose single mutations were previously validated to significantly enhance binding; green, double and single mutants of the mutations in ACE2 2 .v2.4.

Article Snippet: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 g , 60 s), washed with ice-cold Dulbecco′s phosphate-buffered saline (PBS) containing 0.2% of bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described ) and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS–BSA.

Techniques: Expressing, Mutagenesis, Fluorescence, Flow Cytometry, Binding Assay

Journal: Cell reports

Article Title: KRAS G12C -independent feedback activation of wild-type RAS constrains KRAS G12C inhibitor efficacy

doi: 10.1016/j.celrep.2022.110993

Figure Lengend Snippet: (A) KRAS- G12C mutant cell lines were treated with AMG 510 (100 nM) for 0, 4, 24, 48, and 72 h. Blot analysis was performed for phospho- (p)MEK, pERK, pRSK, pAKT, and total MYC with GAPDH as a loading control. (B) Densitometry of pERK normalized to GAPDH for blots in (A) and cell lines treated with ARS-1620 (10 μM) or MRTX849 (100 nM) for 4, 24, 48, and 72 h; results represent an average of pERK across all eight cell lines). (C and G) Cell lines were treated with 10 μM ARS-1620 or 100 nM AMG 510 for 4, 24, 48, or 72 h either refreshed at each time point or not refreshed throughout the time course, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (D and H) Densitometry of pERK normalized to GAPDH for blots in (C) and (G). (E and I) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (C) and (G). (F and J) LC/MS analysis of ARS-1620 (10 μM) or AMG 510 (100 nM) drug levels in media over time incubated either alone at 37°C or with the H358 cell line.

Article Snippet: After indicated inhibitor treatment, RAS activity was assessed by GST-RAF-RBD pulldown (Cell Signaling Technologies), followed by immunoblotting with pan-RAS or RAS isoform–specific antibodies.

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: Cell reports

Article Title: KRAS G12C -independent feedback activation of wild-type RAS constrains KRAS G12C inhibitor efficacy

doi: 10.1016/j.celrep.2022.110993

Figure Lengend Snippet: (A) SW1463, MIA PaCa-2, and H358 cell lines were treated with 1 or 10 μM ARS-1620 or 0.1 or 0.3 μM AMG 510 for 4 h or 7 days with drug refreshed every 2 days, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (B) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (A). (C) Densitometry analysis of KRAS-, NRAS-, and HRAS-GTP levels normalized to input KRAS and GAPDH loading control for blots in (A). (D and E) Densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of cell lines treated with AMG 510 (100 nM) for 4, 24, 48, or 72 h in . (F and H) SW1463, MIA PaCa-2, and H358 cell lines were subject to siRNA knockdown of NRAS, HRAS, and NRAS and HRAS and treated with AMG 510 (100 nM) or RM-018 (100 nM) for 24, 48, and 72 h. Blot analysis was performed for pMEK, pERK, pRSK, pAKT, and total NRAS, HRAS, KRAS, and MYC with GAPDH as a loading control. (G and I) Densitometry of pERK normalized to GAPDH for blots in (F) and (H); results represent an average of pERK across three cell lines. Statistical significance was evaluated by Student’s t test, where *p < 0.05 and **p < 0.01. ns, not significant.

Article Snippet: After indicated inhibitor treatment, RAS activity was assessed by GST-RAF-RBD pulldown (Cell Signaling Technologies), followed by immunoblotting with pan-RAS or RAS isoform–specific antibodies.

Techniques:

Journal: Cell reports

Article Title: KRAS G12C -independent feedback activation of wild-type RAS constrains KRAS G12C inhibitor efficacy

doi: 10.1016/j.celrep.2022.110993

Figure Lengend Snippet: (A) MIA PaCa-2 cells were treated with AMG 510 (100 nM) or RM-018 (100 nM) alone or in combination with the SHP2 inhibitor RMC-4550 (1 μM) for 4, 24, 48, or 72 h, and lysates were subject to a RAF-RBD pull-down and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. (B) Densitometry analysis of KRAS-GTP levels normalized to input KRAS and GAPDH loading control (bar) for blots and pERK normalized to GAPDH loading control (line) in (A). Data represent combined densitometry for MIA PaCa-2 in (A) and SW1463 and H358 in . (C) Densitometry analysis of KRAS-GTP levels to input KRAS and GAPDH loading control and densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of cell lines treated with AMG 510 alone or in combination with RMC-4550 or the MEK inhibitor trametinib (10 nM) in . (D and E) Densitometry analysis of pERK normalized to loading control GAPDH for blots of KRAS12C mutant non-CRC and CRC subjected to indicated treatments in in and . (F and G) Densitometry analysis of KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots of KRAS G12C -mutant CRC cell lines treated with AMG 510 alone or in combination with RMC-4550 or the EGFR inhibitor panitumumab (30 μg/mL) for 4 or 48 h in . (H) Quantification of crystal violet stain of CRC cell lines treated with AMG 510 (100 nM), RMC-4550 (1 μM), panitumumab (30 μg/mL), trametinib (10 nM), or a combination for 10–14 days in .

Article Snippet: After indicated inhibitor treatment, RAS activity was assessed by GST-RAF-RBD pulldown (Cell Signaling Technologies), followed by immunoblotting with pan-RAS or RAS isoform–specific antibodies.

Techniques: Mutagenesis, Staining

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) Structures of thiopurines used this study in comparison to guanine. (B) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.1 then treated with 6-thioguanine (6-TG), 6-thioguanosine (6-TGo), or 6-mercaptopurine (6-MP). Supernatants were harvested after 48 h and stored at -80°C until titering on Vero’76 cells. Mock-infected cells were similarly treated with 6-TG, 6-TGo, 6-MP, or DMSO vehicle control for 48 h before testing cell viability with CellTiter 96 AQueous One (n = 3 ± SEM). Dotted line indicates Limit of Detection. (C) Summary table of 50% Cytoxic Concentration (CC 50 ), 50% Effective Concentration (EC 50 ), and Selectivity Index (SI) calculated for (A-C). (D) AlamarBlue cell viability assay of hTert-BJ, HCT-8, and Huh-7.5 cells treated with 6-TG (n = 3±SEM). (E-H) TCID50 assays for (E) HCoV-OC43 infected HCT-8 cells and (F) HCoV-229E infected Huh-7.5 cells. Cells were infected with an MOI of 0.1 then treated with tunicamycin (Tm), 6-TG, 6-TGo, 6-MP, or DMSO (n≥3 ± SEM, statistical significance was determined by one-way ANOVA). hTERT-BJ cells were infected with HCoV-OC43 (G) or HCoV-229E (H) at an MOI of 0.1 and treated with 6-TG, Tm, or DMSO. Supernatants were harvested after 23 h and stored at -80°C before titering on BHK-21 or Huh7.5 (n = 3–4 ± SEM, statistical significance was determined by one-way ANOVA). LOD = Limit of Detection for virus titer. (*, p<0.05; **, p<0.01; ns, non-significant).

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Infection, Concentration Assay, Viability Assay

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 7, then treated with 6-TG or DMSO. Lysates were harvested 48 h after infection and were probed by western blotting as indicated. Concentrations in legend are in μM. (B) Huh-7.5 cells were infected with SARS-CoV-2 at an MOI of 5, then treated with 6-TG or DMSO. Lysates were harvested 48 h after infection and were probed by western blotting as indicated. Concentrations in legend are in μM. (C) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike (S), Membrane (M), Envelope (E), Nucleoprotein (N) with a C-terminal 2xStrep tag, or empty vector (EV) then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection and probed by western blotting as indicated. (D) 293T cells were transfected with SARS-CoV-2 Spike or EV then treated with 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. Concentrations in legend are in μM. (E) AlamarBlue cell viability assay of 6-TG treated 293T cells (n = 3±SEM). (F) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike, a Spike mutant lacking the Furin cleavage site between S1 and S2 (S0), or EV then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 24 h after transfection, treated with PNGase F to remove N-linked glycans, and probed by western blotting as indicated. (G) as in (C) except 293T cells were transfected with plasmids encoding C-terminal FLAG-tagged Spike (S-FL) or HCoV-MERS Spike (MERS). (H) as in (C) except 293T cells were transfected with plasmids encoding FLAG-tagged Spike from HCoV-OC43 (OC43) or HCoV-HKU1 (HKU1).

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Infection, Western Blot, Transfection, Plasmid Preparation, Viability Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) AlamarBlue cell viability assay of 293T cells treated with brefeldin A (BFA) (n = 4±SEM). (B) 293T cells were transfected with plasmid encoding Gaussia luciferase for 18 h then treated with 6-TG, BFA or DMSO vehicle control for 6 h. Supernatants were recovered from the cells and measured for luciferase activity. (C) 293T cells were transfected with plasmids encoding firefly luciferase and Gaussia luciferase then treated with 6-TG, BFA, or DMSO. After 24 h, supernatants were removed and analyzed as in (B). Cell lysate was harvested in Reporter Lysis Buffer and stored at -80°C until luciferase and Gaussia luciferase activities were measured. (For B and C, n = 3 ± SEM, statistical significance was determined by paired t-test compared to DMSO-treated cells; *, p<0.05; **, p<0.01; ***, p<0.001; ns, non-significant). (D) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike or EV then treated with 6-TG, BFA, or DMSO. Lysates were harvested 24 h after transfection in 2x Laemmli buffer and were probed by western blotting as indicated. (E) 293T cells were co-transfected with plasmids encoding EGFP and either Spike (S), Spike with 19 residue C-terminal truncation (SΔ19), or EV and then treated with 10 μM 6-TG or DMSO. After 24 h, cells were harvested, surface-stained for Spike then fixed prior to analysis by flow cytometry. EGFP+ cells were gated for analysis of the number of Spike+ cells and Median Fluorescent Intensity (MFI) (n = 4±SEM statistical significance was determined by paired t-test between 6-TG and DMSO treated cells; *, p<0.05).

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Viability Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Lysis, Western Blot, Staining, Flow Cytometry

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) 293T cells were transfected with equal quantities of SARS-CoV-2 S, M, E, and N plasmids or empty vector (EV) then treated with 10 μM 6-TG or DMSO vehicle control. Lysates were harvested 48 h after transfection and probed by western blotting as indicated. (B) Virus-like particles from supernatants of cells transfected in (A) were concentrated by ultracentrifugation. Samples from two independent VLP preparations were probed by western blotting as indicated. (C) 293T cells were transfected with plasmids encoding S, E, M, and N in a 1:2:2:1 ratio, substituting one of the structural proteins for EV as indicated, treated with 6-TG or DMSO then processed as in (A). (D) SARS-CoV-2 Spike pseudotyped, luciferase-expressing lentivirus particles were concentrated by ultracentrifugation. Samples from three independent lentivirus preparations were probed by western blotting as indicated. (E) Genomes from three independent lentivirus preparations were quantified by RT-qPCR (n = 3, statistical significance was determined by paired t-test; ns, non-significant). (F) 293A cells stably expressing ACE2 or empty vector control were transduced with lentivirus from three independent preparations. After 24 h, lysates were harvested and measured for luciferase activity (n = 3, statistical significance was determined by two-way ANOVA; *, p<0.05; ns, non-significant). (G) 293A cells were infected HCoV-OC43 at an MOI of 0.1, then treated with 6-TG or DMSO. Supernatants were concentrated as in (D) before virions were fixed and imaged by TEM with negative staining. Five virions from both 6-TG- and DMSO-treated samples are shown at 150,000 X magnification. Scale bar = 100 nm. Arrowhead indicates examples of Spike proteins extending from virion.

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Transfection, Plasmid Preparation, Western Blot, Luciferase, Expressing, Quantitative RT-PCR, Stable Transfection, Transduction, Activity Assay, Infection, Negative Staining

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) HPRT1 catalyses a reaction between 6-TG and phosphoribosyl diphosphate (PRPP) to generate 6-thioguanosine monophosphate (6-TGMP) and pyrophosphate products. 6-TG methylated at the N9 nitrogen (6-TG-Me) was designed to be resistant to processing by HPRT1. (B) 293T cells were infected with HCoV-OC43 at an MOI of 0.1 then treated with 6-TG or 6-TG-Me. Supernatants were harvested at 24 hpi and stored at -80°C until titering on BHK-21 cells (n = 3 statistical significance was determined by paired ratio t-test; *, p<0.05; **, p<0.01; ns, non-significant). (C) 293T cells were transfected with SARS-CoV-2 Spike vector or an empty vector control then treated with 6-TG, 6-TG-Me, or DMSO vehicle control. Lysates were harvested 24 h after transfection and probed by western blotting as indicated. (D) 293T cells (parental), non-targeting (NT) CRISPR control cells, or two independent CRISPR-edited HPRT1 knockout cell lines (HPRT1-KO1 and -KO2) were infected with HCoV-OC43 at an MOI of 0.1 for 1 h prior to treatment with DMSO or 10 μM 6-TG for the remaining 23 h. Lysates were prepared 24 h and analyzed by western blotting as indicated. (E) As in (D) but cell supernatants were harvested at 24 h and titered as in (B) (n = 6 ±SEM, statistical significance was determined by paired ratio t test; ***, p<0.001; ****, p<0.0001; ns, non-significant, LOD = Limit of Detection.). (F) The cell lines in (D) were transfected with plasmids encoding codon-optimized HCoV-OC43-Spike or an empty vector followed by treatment with DMSO or 10 μM 6-TG. Lysates were prepared at 24 h and analyzed by western blotting as indicated.

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Methylation, Infection, Transfection, Plasmid Preparation, Western Blot, CRISPR, Knock-Out

Journal: PLoS Pathogens

Article Title: Thiopurines inhibit coronavirus Spike protein processing and incorporation into progeny virions

doi: 10.1371/journal.ppat.1010832

Figure Lengend Snippet: (A) AlamarBlue cell viability assay of 293T cells treated Rac1 Inhibitor V (Rac1iV), Rhosin, CASIN, or ML099 (n = 3±SEM). (B) Summary table of 50% cytotoxic concentration (CC 50 ) and inhibitor target of compounds tested in (A). (C) 293T cells were transfected with SARS-CoV-2 Spike vector or an empty vector control (EV) then treated with 6-TG, GTPase inhibitors, or DMSO vehicle control. Concentrations in legend are in μM. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. (D) 293T cells were transfected with plasmids encoding SARS-CoV-2 Spike or EV control then co-treated with 6-TG, 100 μM ML099 GTPase agonist, or DMSO. Lysates were harvested 24 h after transfection and were probed by western blotting as indicated. Concentrations in legend are in μM. (E) as in (D) except some samples were pre-treated (Pre) for 4 h with ML099. (F) HCT-8 and 293A were infected with an MOI of ~0.1 then treated with 10 μM 6-TG and/or 100 μM ML099 or DMSO vehicle control. After 24 h, the supernatants were harvested and stored at -80°C until titering on BHK-21 (n = 3 ± SEM, statistical significance was determined by one-way ANOVA; *, p<0.05; **, p<0.01; ns, non-significant). LOD = Limit of Detection for virus titer.

Article Snippet: Membranes were blocked with 5% bovine serum albumin in tris-buffered saline/0.1% [vol/vol] tween-20 (TBS-T) before probing overnight at 4°C with antibodies raised to the following targets: mouse anti-puromycin (Millipore-Sigma, MABE343), mouse anti-OC43 N (CoV antibody, OC43 strain, clone 541-8F, Millipore-Sigma, MAB9012), rabbit anti-OC43 S (Cusabio, CSB-PA336163EA-1HIY), rabbit anti-BiP (Cell Signaling Technologies (CST, #3177), mouse anti-XBP1s (CST, #12782), mouse anti-CHOP (CST, #2895), rabbit anti-α-Tubulin (CST, #2125), rabbit anti-SARS-CoV-2 S1 RBD (Elabscience, E-AB-V1006), rabbit anti-SARS-CoV-2 E (abbexa, abx226552), rabbit anti-SARS-CoV-2 M (Novus Biologicals, NBP3-05698), rabbit anti-SARS-CoV-2 N (Novus Biologicals, NBP3-05730), mouse anti-DYKDDDDK (CST, #8146), mouse anti-HIV-p24 (Abcam, ab9071) mouse anti-Strep tag (IBA, 2-1507-001), mouse anti-HPRT (Santa Cruz, sc-376938), rabbit anti-β-actin (HRP-conjugate; CST, #5125), and rabbit anti-β-actin (CST, #4967).

Techniques: Viability Assay, Concentration Assay, Transfection, Plasmid Preparation, Western Blot, Infection