Journal: bioRxiv

Article Title: A stable subgenomic reporter coronavirus enables transcriptional profiling of bystander cells

doi: 10.64898/2026.02.27.708290

Figure Lengend Snippet: Reagents for analysing HCoV-OC43. A. Western blot of Mv.1.Lu cells infected with HCoV-OC43 at an MOI of 10 PFU/cell and harvested at the indicated time points, probed with two anti-Nucleocapsid (N) antibodies, derived from Sheep (Sh) polyclonal antiserum from the MRC PPU & CVR Coronavirus toolkit and a commercial Rabbit (Rb) polyclonal antiserum. Host HSP90 is included as a loading control. B . Immunofluorescence microscopy of HCoV-infected A549 cells, stained with Sheep anti-N and anti double-stranded RNA (dsRNA), a marker of RNA replication. C . Immunoprecipitation (IP) of lysates from HCoV-OC43-infected cells (MOI 3, 24 hpi) using Sheep anti-N and probed with Sheep anti-N and anti-Spike (S). Host HSP90 is included as a loading control. D . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells, stained with mouse monoclonal (Mm) or rabbit polyclonal (Rb) anti-S. E . Silver stain (SS) and western blotting (WB) of virions purified by ultracentrifugation through a 30% sucrose cushion. The supernatant (sup) before and after sucrose cushion, and the pellet, containing purified virions, are shown. Viral structural proteins are indicated by red arrowheads and BSA from cell culture medium by a red asterisk. F . Immunofluorescence microscopy of HCoV-OC43-infected A549 cells stained with anti-SARS-CoV M. Scale bars represent 10 µm.

Article Snippet: Membranes were probed with sheep polyclonal anti-Nucleocapsid anti-serum (MRC PPU & CVR Coronavirus Toolkit, Sheep No. DA116, 1:1000) , rabbit anti-Nucleocapsid (40643-T62, Sino Biological, 1:1000), anti-Spike (PAB21478-100, The Native Antigen Company, 1:1000), anti-SARS-CoV Membrane (ABIN1887462, Antibodies Online, 1:200), anti-MHV nucleocapsid J3.3 ( ) (1:200), anti-GAPDH (AM4300, Invitrogen, 1:4000) and anti-HSP90 (MA5-35624, Invitrogen, 1:2000).

Techniques: Western Blot, Infection, Derivative Assay, Control, Immunofluorescence, Microscopy, Staining, Marker, Immunoprecipitation, Silver Staining, Purification, Cell Culture

Journal: Scientific Reports

Article Title: The von Willebrand factor stamps plasmatic extracellular vesicles from glioblastoma patients

doi: 10.1038/s41598-021-02254-7

Figure Lengend Snippet: VWF is transported in circulating EVs from GBM patients. ( A ) Equal volume of protein lysates from EV fractions, obtained with SEC followed by ultracentrifugation, and their corresponding plasma, from one GBM patient and one healthy donor, were separated by SDS-PAGE and analyzed by immunoblot for VWF, FCN3, and HSP70. Blots are representative of n = 3. ( B ) mRNA expression of von Willebrand factor (VWF) and ficolin-3 (FCN3) in healthy subjects and GBM patients estimated from The Cancer Genome Atlas (TCGA, n > 500 for GBM patients). ( C ) Anomalies currently reported in VWF and FCN3 genes. ( D ) Equal volume of protein lysates from EV fractions (3 GBM and 3 Healthy) were separated by SDS-PAGE and analyzed by immunoblot for VWF, GM130 (putative intracellular membrane protein contaminant), and Apolipoprotein A1 (APOA1, plasmatic protein). Healthy donor plasma and total cell lysate from GBM cells serve as controls. Blots are representative of n > 7 individual samples. ( E ) Densitometric analysis was performed on 7 and 8 independent EV preparations from Healthy and GBM liquid biopsies, respectively. ( F ) Concentration of VWF in plasmatic EVs from healthy donors and GBM patients (n = 20). Mann–Whitney test, * p < 0.05, ** p < 0.01, *** p < 0.001. S.D. are shown in panel E and S.E.M. in panel F.

Article Snippet: EVs were separated and concentrated from 500 μl of plasma as described above and the pellet was directly lysed in boiling Laemmli for 10 min. Proteins were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blotted with the following antibodies, all diluted at 1/1000: CD9 (EXOAB-CD9A-1, SBI), CD63 (EXOAB-CD63A-1, SBI), VWF (sc-53466, Santa Cruz), FCN3 (CL7767AP, Cedarlane) , Apolipoprotein A1 (sc-376818, Santa Cruz), HSP70 (EXOAB-HSP70A, SBI), and GM130 (ab52649, Abcam) diluted at 1/1000.

Techniques: Clinical Proteomics, SDS Page, Western Blot, Expressing, Membrane, Concentration Assay, MANN-WHITNEY

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: The antiviral effect of Tg against HCoV-229E infection does not require IRE1, ATF6, or PERK expression. (A) Stable UPR knockdown A549 cell lines were generated via lentiviral transduction. Silencing of protein expression was confirmed via western blot. (B–F) A549 shCTRL, shIRE1, shATF6, and shPERK cells were primed with DMSO or Tg (0.05 µM) for 30 minutes, then washed prior to infection with HCoV-229E (MOI 0.05). Cell lysates (B–E) or supernatants (F) were collected at 24 hpi. (B–D) Protein expression of IRE1, ATF6, PERK and HCoV-229E N were assessed by western blot. (E) Changes in gene expression were quantified by RT-qPCR. Data are normalized to actin and set relative to shCTRL DMSO. (F) Supernatants were used to determine viral titers by plaque assay on Huh7 cells. Due to variability between replicates, titration data is expressed as a percentage relative to the shCTRL DMSO in each experiment. Graphs show means ± SD from 3 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Infection, Expressing, Knockdown, Generated, Transduction, Western Blot, Gene Expression, Quantitative RT-PCR, Plaque Assay, Titration

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological inhibition of IRE1 or ATF6 does not affect the antiviral activity of Tg against HCoV-229E infection. (A and B) A549 cells were primed with DMSO or Tg (0.05 µM) in the absence or presence of KIRA6 (10 µM) or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hours post-treatment. Xbp1s (A) or HERPUD1 (B) protein expression was assessed by western blot. (C) A549 cells were treated with DMSO, KIRA6 (10 µM), or Ceapin-A7 (6 µM) for 24 hours. Alamar blue assay was used to assess cell viability, which is expressed as a percentage relative to DMSO. (D and F) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or KIRA6 (10 µM). Cell lysates were collected at 24 hpi to assess XBP1s and N gene expression by RT-qPCR (D and F), or Xbp1s and HCoV-229E N protein expression by western blot (E). (G and I) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of Ceapin-A7 (6 µM), then washed prior to infection with HCoV-229E (MOI 0.05). Viral inoculum was removed 2 hours later and replaced with media containing DMSO or Ceapin-A7 (6 µM). Cell lysates were collected at 24 hpi to assess HERPUD1 and N expression by RT-qPCR (G and I), or HERPUD1 and HCoV-229E N protein expression by western blot (H). RT-qPCR data are normalized to actin and set relative to DMSO. (J) A549 cells were primed with DMSO or Tg (0.05 µM) in the presence or absence of KIRA6 (10 µM) or Ceapin-A7 (6 µM), alone or in combination, prior to infection with HCoV-229E (MOI 0.05). Inoculum was removed 2 hours later and replaced with media containing the respective inhibitor(s). Viral supernatants were collected 24 hpi and titrated by plaque assay on Huh7 cells. Graphs show mean ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate (* p < 0.05, *** p < 0.001; **** p < 0.0001). Representative western blots are shown.

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Inhibition, Activity Assay, Infection, Expressing, Western Blot, Alamar Blue Assay, Gene Expression, Quantitative RT-PCR, Plaque Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: Pharmacological activation of PERK inhibits HCoV-229E replication. (A–C) A549 cells were treated with DMSO or one of the selective UPR activators for 24 hours. IXA4 (10 µM) activates IRE1, AA147 (10 µM) activates ATF6, and CCT020312 (5 µM) activates PERK. RNA lysates were collected 24 hours post-treatment and the expression of genes downstream of each UPR pathway was assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (D) A549 cells were primed with Tg (0.5 µM) for 30 minutes, then washed and infected with HCoV-229E (MOI 0.5). Alternatively, cells were treated with AA147 (10 µM) or CCT020312 (5 µM) for 24 hpi. Cell lysates were collected to assess the expression of HERPUD1, PERK and 229E N protein by western blot. (F and G) A549 cells were primed with DMSO or Tg (0.05 µM) for 30 minutes prior to infection with HCoV-229E (MOI 0.05). Alternatively, cells were infected and then treated with media containing DMSO or the selective UPR activators alone or in combination. Cell lysates and supernatants were collected at 24 hpi. (F) Changes in HCoV-229E N gene expression were assessed by RT-qPCR. Data are normalized to actin and set relative to DMSO. (F) Viral titer was assessed by plaque assay on Huh7 cells. (H–J) A549 cells were infected with HCoV-229E (MOI 0.05), then incubated with increasing concentrations of CCT020312 for 24 h. Cell lysates were collected to assess changes in HCoV-229E N gene (H) and CHOP (I) expression by RT-qPCR. (J) Cell viability was assessed by Alamar blue assay. Graphs show means ± SD from 2–3 independent experiments performed in triplicate, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way ANOVA or t -test (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Infection, Western Blot, Gene Expression, Plaque Assay, Incubation, Alamar Blue Assay

Journal: RSC Chemical Biology

Article Title: Chemical modulation of the unfolded protein response reveals an antiviral role for the PERK pathway in human coronavirus 229E infection

doi: 10.1039/d5cb00242g

Figure Lengend Snippet: UPR activation by tunicamycin does not recapitulate the antiviral effect of Tg. (A–C) A549 cells were primed with Tg (0.5 µM) for 30 minutes or with Tm (1 µg mL −1 ) for 4 hours. Cells were then washed and either mock-infected or infected with HCoV-229E (MOI 0.05). At 24 hpi, cell lysates were collected to evaluate expression of UPR target genes ( CHOP , HERPUD1 and Xbp1s ) or HCoV-229E N gene by qPCR. Graphs show means ± SD from 2 independent experiments, with qPCR performed in technical triplicate. Statistical significance was assessed by one-way or two-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001; **** p < 0.0001).

Article Snippet: Primary antibodies against IRE1α (Cell Signaling Technology (CST) 3294, dilution 1 : 1000), ATF6 (CST 8089, dilution 1 : 1000), PERK (CST 5683, dilution 1 : 1000), XBP1s (CST 12782T, dilution 1 : 1000), HERPUD1 (Abcam ab150424, dilution 1 : 1000), HCoV-229E N (SinoBiological 40640-T62, dilution 1 : 10 000), beta-actin (Abcam ab8226, dilution 1 : 5000), alpha-tubulin (Thermo Scientific MA5-31466, dilution 1 : 5000), and GAPDH (Thermo Scientific MA5-15738, dilution 1 : 1000) were used for western blotting.

Techniques: Activation Assay, Infection, Expressing