Journal: eLife

Article Title: Controlling motor neurons of every muscle for fly proboscis reaching

doi: 10.7554/eLife.54978

Figure Lengend Snippet:

Article Snippet: Antibody , rat mAb anti-FLAG , Novus Biologicals , Cat# NBP1-06712; RRID: AB_1625981 , (1:200) , .

Techniques: Muscles, Construct, Software

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) An overview of the subunit architecture of human condensin I, with a table of equivalent subunits in the condensin and cohesin complexes. ( B ) ATPase assay of human condensin I pentamer (CI), tetramers lacking HAWK domains and reconstituted pentamers. Q refers to complexes with a mutation in the Q-loop, which cannot bind ATP. The lower panel shows SDS-PAGE analysis of the ATPase reaction mixture after completion of the assay. Purine nucleoside phosphorylase (PNP) enzyme used in the ATPase assay is indicated as a loading control. Experimental sample sizes for each lane: 1. n = 9, 2. n = 8, 3. n = 6, 4–10. n = 4, and 11–12. n = 3, where each technical replicate was performed with a distinct protein aliquot at a different time. P values determined using unpaired two-tailed t tests with Welch’s correction to account for unequal variance. ( C ) Fold-stimulation of the ATPase rate when 50 bp dsDNA is titrated, determined by dividing the ATPase rate at each DNA concentration, by the rate without DNA, for each DNA concentration, n = 3 technical replicates were performed with a distinct protein aliquot. ( D ) Gel shift assay of condensin I pentamer and tetramers, respectively, binding to 50 bp dsDNA labelled with Cy5. In each case, column value and error bars indicate, mean and s.e.m., respectively. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: ATPase Assay, Mutagenesis, SDS Page, Control, Two Tailed Test, Concentration Assay, Gel Shift, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Comparison of two different batches of recombinant CIWT pentamer, and CIΔG and CIΔD2 tetramers. Trend of tetrameric complexes being significantly more active than pentameric condensin I is consistent. Data from three technical replicates. Error bars represent s.e.m. P values indicated are from unpaired, two-tailed t test with Welch’s correction. ( B ) Mass photometry data of condensin I tetramers, CIΔG, with and without NCAPG being added to reconstitute the pentamer. ( C ) Mass photometry data of condensin I tetramers, CIΔD2, with and without NCAPD2 being added to reconstitute the pentamer. ( D – F ) Mass photometry data of condensin I pentamers, CID2ΔC, CIHΔN and CIHΔN,D2ΔC, respectively, consistent with pentameric mass and confirming they are the major species present. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Comparison, Recombinant, Two Tailed Test

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) ATPase assay of condensin I mutants, with mutations illustrated on the right. Mutation CIΔG indicate tetrameric condensin I lacking NCAPG. CIΔSB indicates deletion of the NCAPH region where NCAPG binds. Gp1 and Gp2 indicates mutations in NCAPG homologous to patch1 and patch2 where Ycg1 and SMC2 interact in yeast condensin. CIGΔC indicates deletion of NCAPG C-terminal disordered region, CINΔH indicates deletion of NCAPH N-terminal disordered region and CID2ΔC indicates deletion of NCAPD2 C-terminal disordered region. CIQHΔN, CIQD2ΔC and CIQHΔN,D2ΔC indicating ATPase dead Q-loop mutant controls. The lower panel is SDS page analysis of the ATPase reaction mixture after completion of the assay. Purine nucleoside phosphorylase (PNP) enzyme used in the ATPase assay is indicated as a loading control. Experimental sample size for each condition: 1. n = 17, 2–3. n = 8, 4–5. n = 5, 6–9. n = 4, 10. n = 6, 11–13. n = 5, 14. n = 3, 15. n = 4, 16. n = 5 and 17. n = 3, where each technical replicate was performed using a distinct protein aliquot, collected at a different time. P values shown are comparisons to CIWT, determined using unpaired two-tail t tests with Welch’s correction to account for unequal variance. In each case, column value and error bars indicate mean and s.e.m., respectively. ( B ) AlphaFold2 model of NCAPG with the C-terminal end of NCAPD2, with NCAPD2 coloured with AlphaFold2 (Jumper et al, ; preprint: Evans et al, ) pLDDT confidence score. ( C ) Surface charge of the NCAPG/D2 interface. ( D ) Fluorescence polarisation binding assay of 5-FAM NCAPD2 1376-1398 . Error bars indicate mean ± s.d., data from three technical replicates, performed with distinct protein aliquots, read three times. Solid line indicates data fit to determine Kd. “NCAPGap” indicates NCAPG with acid patch mutation (D136K, D137K, D141K). ( E – G ) Equivalent data as ( B – D ) for NCAPH. Fluorescence polarisation in ( G ) was performed with 5-FAM NCAPH 55-77 . .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: ATPase Assay, Mutagenesis, SDS Page, Control, Fluorescence, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) AlphaFold2 model of NCAPG with the C-terminal end of KIF4A, with KIF4A coloured with AlphaFold2 (Jumper et al, ; preprint: Evans et al, ) pLDDT confidence score. ( B ) Surface charge of the NCAPG/KIF4A interface. ( C ) Overlay of NCAPG showing binding of KIF4A peptide (blue) overlaps with binding of NCAPD2 and NCAPH (pink and green, respectively). ( D ) Fluorescence polarisation binding assay of 5-FAM labelled KIF4A. “NCAPGap” indicates NCAPG with acid patch mutation (D136K, D137K, D141K). Solid line indicates Kd fit, Kd value shown ± standard error. ( E ) Competition FP assay with 100 nM 5-FAM NCAPD2 1376-1398 , 6 μM NCAPG with 10 μM KIF4A, KIF4A 1206-1228 , WT or Mut (mutant) peptide. ( F ) Competition FP assay with 100 nM 5-FAM NCAPH 55-77 , 4 μM CINΔH,D3ΔC with 100 μM KIF4A, KIF4A 1206-1228 , WT or Mut peptide. For FP data in ( D – F ), error bars indicate mean ± s.d., and data were derived from three technical replicates performed with distinct protein aliquots, read three times. P values determined using unpaired, two-tailed t tests. ( G ) ATPase assay of WT and pentameric condensin I lacking the C-terminal region of NCAPD2 and/or the N-terminal region of NCAPH, in the presence and absence of 50 μM KIF4A 1206-1228 , WT or Mut peptide. ATPase assays from 3 repeats, error bars indicate s.e.m., P values determined with unpaired, two-tailed t test with Welch’s correction. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Binding Assay, Fluorescence, Mutagenesis, FP Assay, Derivative Assay, Two Tailed Test, ATPase Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Schematic of the single-molecule loop extrusion assay under constant buffer flow and corresponding snapshots of a loop formed by CIWT in the presence of KIF4A 1206-1228 peptide. ( B ) Fraction of DNA strands with at least one looping event during a 1000 s acquisition time, in the presence of 1 nM human CIWT and CIΔG ( n = 4 and 10 experiments, respectively). ( C ) Fraction of DNA strands with at least one looping event during a 1000 s acquisition time, in the presence of 0.5 nM condensin I WT and WT supplied with 1000x KIF4A 1206-1228 peptide ( n = 3 experiments). ( B , C ) Mean ± s.d. is shown, the total number of DNA tethers analysed is indicated by n tot and P values calculated with Wilcoxon rank-sum tests. ( D ) Example snapshots, the corresponding ( E ) kymograph and ( F ) time trace of DNA lengths for a loop extrusion event by CIΔG without flow. ( G ) Box-whisker-plots showing the loop extrusion rates of CIWT, CIΔG and CIWT in the presence of KIF4A 1206-1228 peptide. Calculated from n tot = 55 looping events, from replicates detailed in ( B , C ). For the box plots, the central line denotes the median, the box limit denotes the 25th–75th percentile and the whiskers denote minimum and maxima. The P values were calculated using Welch’s t test. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Whisker Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A ) Current models (Shaltiel et al, ; Dekker et al, ) for DNA loop extrusion by condensin complexes require loops to be initiated by DNA being “threaded” through the NCAPH Kleisin. ( B ) In the presence of interactions between NCAPD2/H and NCAPG, the threading step could be blocked. ( C ) The interaction with KIF4A could allow DNA threading and loop initiation. ( D ) Loss of NCAPG could also reduce inhibition of DNA threading, but may result in reduced DNA-binding affinity and slippage of NCAPG/H DNA anchor.

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Inhibition, Binding Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: ( A , B ), AlphaFold2 model of Ycg1 with Lrs4 and Sgo, respectively, with insert of electrostatic potential. ( C ) Sequence alignment of SLiMs that bind the equivalent site of NCAPG and Ycg1. ( D , E ) Fluorescence polarisation binding data of Lrs4 and Sgo peptides with yeast condensin complex. Error bars indicate mean ± s.d., data from three technical replicates performed with distinct protein aliquots, read three times. ( F ) A general model of the role SLiM HAWK association has in regulating condensin and cohesin activity. .

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Sequencing, Fluorescence, Binding Assay, Activity Assay

Journal: The EMBO Journal

Article Title: Molecular mechanism of condensin I activation by KIF4A

doi: 10.1038/s44318-024-00340-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cells pellets were lysed in human condensin purification buffer (20 mM HEPES pH 8, 300 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 10% glycerol) supplemented with 1 Pierce protease inhibitor EDTA- free tablet (Thermo Scientific) or 1 cOmplete protease inhibitor EDTA-free tablet (Roche) per 50 mL and 25 U/ml of Benzonase (Sigma) with a Dounce homogenizer followed by brief sonication.

Techniques: Recombinant, Residue, Mutagenesis, Sequencing, Software

Journal:

Article Title: Solid-phase extraction of N -linked glycopeptides

doi: 10.1038/nprot.2007.42

Figure Lengend Snippet: Schematic diagram of solid-phase extraction of N-linked glycopeptides. Proteins are first proteolyzed into peptides. Glycosylated peptides are then oxidized and coupled to a solid support. Non-glycopeptides are removed by successive washes. The amino-terminals of glycopeptides are labeled by succinic anhydride carrying either d0 or d4. N-linked glycopeptides are then released by PNGase F and analyzed by mass spectrometry.

Article Snippet: Make up fresh Denaturing buffer: 8M urea in 0.4M NH 4 HCO 3 with 0.1% SDS Sodium chloride: 1.5 M SDS stock solution: 10% Standard peptide: 1 µM Angiotensin I peptide in water (Sigma-Aldrich #A9650), and 1 µM of Neurotensin peptide in water (Sigma-Aldrich #A6383) Hydrochloric acid (HCl): 5 N Acetic acid: 0.4% PNGase F: New England Biolabs, Ipswich, MA Sequencing grade trypsin: Promega, Madison, WI Acetonitrile (ACN) BCA Protein Assay Kit – Pierce, Rockford, IL Methanol SDS-PAGE Gels 0.1% trifluoroacetic acid (TFA) HPLC solution A: 0.1% formic acid in water HPLC solution B: 0.1% formic acid in acetonitrile (ACN) Succinic-d0-anhydride: Sigma, St. Louis, MO Succinic-d4-anhydride: C/D/N Isotopes, Pointe-Claire, Quebec, CA Isotopic labeling solution: 2 mg/ml of succinic anhydride in Dimethylformamide (DMF)/pyridine/H 2 O=50/10/40 (v/v/v) Software for sequence assignment from tandem mass spectrum: SEQUEST 21 Software for statistical evaluation of peptide sequence assignment: PeptideProphet 22 EQUIPMENT Tube rocker SpeedVac C18 1cc SepPak columns – Waters, Milford, MA Vial: Glass vials with polyethylene snap cap (Waters, Milford, MA) HPLC: HP1100, Agilent Technologies, Santa Clara, CA Sonicator LCQ or LTQ ion-trap mass spectrometer: Thermo Finnigan, San Jose, CA Quadrupole-time-of-flight (ESI-qTOF) mass spectrometer: Waters, Milford, MA Peptide cartridge packed with Magic C18: Michrom Bioresources, Auburn, CA FAMOS autosampler: DIONEX, Sunnyvale, CA Microcapillary HPLC column: 10 cm × 75 µm i.d. packed with Magic C18 resin (5 µm, 100Å, Michrom Bioresources, Auburn, CA) EQUIPMENT SETUP LC-MS-MS/MS: Peptides are injected into a peptide cartridge packed with Magic C18 using a FAMOS autosampler, and then pass through a 10 cm × 75 µm i.d. microcapillary HPLC column packed with Magic C18 resin.

Techniques: Labeling, Mass Spectrometry

Journal: Frontiers in Microbiology

Article Title: The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

doi: 10.3389/fmicb.2023.1206951

Figure Lengend Snippet: Antiviral activity of nitazoxanide against human seasonal coronaviruses. (A) Schematic representation of genome structure, classification and receptors of the human coronaviruses HCoV-229E, HCoV-OC43 and HCoV-NL63. ORF1a and ORF1b are represented as yellow boxes; genes encoding structural proteins spike (S), nucleocapsid (N), envelope (E), membrane (M), and hemagglutinin-esterase (HE) are shown as blue boxes, and genes encoding accessory proteins are shown as red boxes. hAPN, human aminopeptidase N; 9-O-Ac-Sia, N-acetyl-9-O-acetylneuraminic acid; ACE2, angiotensin-converting enzyme 2. (B–D) MRC-5 (B,C) and LLC-MK2 (D) cells mock-infected or infected with HCoV-229E (B) , HCoV-OC43 (C) or HCoV-NL63 (D) at an MOI of 0.1 TCID 50 /cell were treated with different concentrations of NTZ or vehicle immediately after the adsorption period. In the case of HCoV-NL63, NTZ was removed at 48 h after infection. Virus yield in cell supernatants was determined at 48 (B) , 96 (C) or 120 (D) hours p.i. by infectivity assay (B,C) or RNA quantification by qRT-PCR (D) . Data, expressed as TCID 50 /ml (B,C) or percent of untreated control (D) , represent the mean ± S.D. of duplicate samples. (E) MRC-5 cells infected with HCoV-229E or HCoV-OC43 and LLC-MK2 cells infected with HCoV-NL63 (0.1 TCID 50 /cell) were treated with 3 μM nitazoxanide, the NTZ active metabolite tizoxanide, remdesivir, chloroquine and ribavirin after virus adsorption. In the case of HCoV-NL63, NTZ and tizoxanide were removed at 48 h after infection. Virus yields were determined at 48 (HCoV-229E), 72 (HCoV-OC43) or 120 (HCoV-NL63) hours p.i. by infectivity assay. Data, expressed as TCID 50 /ml, represent the mean ± S.D. of duplicate samples. * p < 0.05; ANOVA test.

Article Snippet: Nitazoxanide [2-acetyloxy- N -(5-nitro-2-thiazolyl) benzamide, Alinia] (NTZ) and tizoxanide (TIZ; Romark Laboratories, L.C.), remdesivir (MedChemExpress), ribavirin and chloroquine (Sigma-Aldrich), dissolved, respectively, in DMSO stock solution (NTZ, TIZ, remdesivir) or water (ribavirin, chloroquine), were diluted in culture medium, added to infected cells after the virus adsorption period, and maintained in the medium for the duration of the experiment, unless differently specified.

Techniques: Activity Assay, Membrane, Infection, Adsorption, Virus, Quantitative RT-PCR, Control

Journal: Frontiers in Microbiology

Article Title: The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

doi: 10.3389/fmicb.2023.1206951

Figure Lengend Snippet: Effect of short treatment with nitazoxanide in OC43 and 229E HCoV-infected human lung cells. (A,B) MRC-5 cell monolayers mock-infected or infected with HCoV-229E and HCoV-OC43 (0.5 TCID 50 /cell) were treated with different concentrations of NTZ or vehicle immediately after the adsorption period. Virus yield (Ο, red line) was determined at 24 h p.i. by infectivity assay. In parallel, cell viability (△, black line) was determined by MTT assay in mock-infected cells (A) . Absorbance (O.D.) of converted dye was measured at λ = 570 nm. IC 50 and LD 50 in μg/ml (B) were calculated using Prism 5.0 software. Data represent the mean ± S.D. of duplicate samples. Selectivity indexes (SI) are indicated. * p < 0.05; ANOVA test.

Article Snippet: Nitazoxanide [2-acetyloxy- N -(5-nitro-2-thiazolyl) benzamide, Alinia] (NTZ) and tizoxanide (TIZ; Romark Laboratories, L.C.), remdesivir (MedChemExpress), ribavirin and chloroquine (Sigma-Aldrich), dissolved, respectively, in DMSO stock solution (NTZ, TIZ, remdesivir) or water (ribavirin, chloroquine), were diluted in culture medium, added to infected cells after the virus adsorption period, and maintained in the medium for the duration of the experiment, unless differently specified.

Techniques: Infection, Adsorption, Virus, MTT Assay, Software

Journal: Frontiers in Microbiology

Article Title: The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

doi: 10.3389/fmicb.2023.1206951

Figure Lengend Snippet: Nitazoxanide acts at postentry level. (A,B) MRC-5 cells mock-infected or infected with HCoV-OC43 (A) or HCoV-229E (B) (0.5 TCID 50 /cell) were treated with NTZ 1 μg/mL [ (A,B) top; filled bars], NTZ 2.5 μg/mL [ (A,B) bottom; filled bars] or vehicle (empty bars) 2 h before infection (Pre), after the adsorption period (Post), or 2 h before infection and treatment was continued during and after the adsorption period (Pre + Post). Virus yield was determined at 24 h p.i. by TCID 50 infectivity assay. (C) MRC-5 cells mock-infected or infected with HCoV-OC43 or HCoV-229E were treated with NTZ (1 μg/mL) or vehicle only during the adsorption period (Ads). Virus yield was determined at 24 h p.i. by infectivity assay. (A–C) Data represent the mean ± S.D. of duplicate samples. * p < 0.01; Student’s t -test. (D) Schematic representation of HCoV genomic RNA transfection assay. (E) MRC-5 cells were transfected with HCoV-OC43 or HCoV-229E genomic RNA for 4 h and treated with NTZ (2.5 μg/mL) or vehicle for 24 h. Virus yield was determined at 24 h after treatment in the supernatant of transfected cells by TCID 50 infectivity assay. Nucleocapsid (N) protein levels in HCoV-OC43 or HCoV-229E RNA-transfected cells are shown (insets). (F) MRC-5 cells infected with HCoV-OC43 were treated with NTZ (1 μg/mL) or vehicle at the indicated times after infection. Virus yield was determined at 24 h p.i. by infectivity assay. (E,F) Data represent the mean ± S.D. of duplicate samples. * p < 0.01; Student’s t -test.

Article Snippet: Nitazoxanide [2-acetyloxy- N -(5-nitro-2-thiazolyl) benzamide, Alinia] (NTZ) and tizoxanide (TIZ; Romark Laboratories, L.C.), remdesivir (MedChemExpress), ribavirin and chloroquine (Sigma-Aldrich), dissolved, respectively, in DMSO stock solution (NTZ, TIZ, remdesivir) or water (ribavirin, chloroquine), were diluted in culture medium, added to infected cells after the virus adsorption period, and maintained in the medium for the duration of the experiment, unless differently specified.

Techniques: Infection, Adsorption, Virus, Transfection

Journal: Frontiers in Microbiology

Article Title: The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

doi: 10.3389/fmicb.2023.1206951

Figure Lengend Snippet: Effect of Nitazoxanide on HCoV-OC43 spike expression. (A,B) MRC-5 cells were mock-infected or infected with HCoV-OC43 at an MOI of 0.5 TCID 50 /cell and treated with different concentrations of NTZ or vehicle immediately after the virus adsorption period. After 24 h, whole cell extracts (WCE) were analyzed for levels of viral S and N proteins by IB using anti-spike and anti-N HCoV-OC43 antibodies [ (A) top], and quantitated by scanning densitometry. Relative amounts of S and N proteins were determined after normalizing to β-actin [ (A) bottom]. The faster-migrating form of the S protein in NTZ-treated cells is indicated by red arrowheads. In the same experiment virus yield was determined at 24 h p.i. in the supernatant of infected cells by TCID 50 infectivity assay (B) . Data represent the mean ± S.D. of duplicate samples. * p < 0.01; ANOVA test. (C) Confocal images of HCoV-OC43 spike glycoprotein (red) in MRC-5 cells mock-infected or infected with HCoV-OC43 at an MOI of 0.5 TCID 50 /cell and treated with NTZ (1 μg/mL) or vehicle for 24 h. Nuclei are stained with Hoechst (blue). Merge images are shown. Scale bar, 20 μm.

Article Snippet: Nitazoxanide [2-acetyloxy- N -(5-nitro-2-thiazolyl) benzamide, Alinia] (NTZ) and tizoxanide (TIZ; Romark Laboratories, L.C.), remdesivir (MedChemExpress), ribavirin and chloroquine (Sigma-Aldrich), dissolved, respectively, in DMSO stock solution (NTZ, TIZ, remdesivir) or water (ribavirin, chloroquine), were diluted in culture medium, added to infected cells after the virus adsorption period, and maintained in the medium for the duration of the experiment, unless differently specified.

Techniques: Expressing, Infection, Virus, Adsorption, Staining

Journal: Frontiers in Microbiology

Article Title: The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein

doi: 10.3389/fmicb.2023.1206951

Figure Lengend Snippet: Nitazoxanide impairs HCoV-OC43 and HCoV-229E spike maturation at an Endo-H sensitive stage. (A–D) MRC-5 cells were mock-infected or infected with HCoV-OC43 (A,C) or HCoV-229E (B,D) at an MOI of 0.5 TCID 50 /cell and treated with NTZ (2.5 μg/mL) or vehicle immediately after the virus adsorption period. After 24 h, WCE were analyzed for levels of S protein by IB using anti-S HCoV-OC43 (A,C) or anti-S HCoV-229E (B,D) antibodies. Red arrowheads indicate the faster-migrating forms of the HCoV-OC43 (A) and HCoV-229E (B) spike proteins in NTZ-treated cells. Spike proteins densitometric and molecular weight analysis (C,D) are shown. ( E ) Diagram of substrate specificity of endoglycosidase-H (Endo-H). Mannose (green circles), N -acetylglucosamine (GlcNAc, blue squares) and asparagine (Asn, red triangle) residues are shown. Scissors represent the cleavage site of Endo-H. (F,G) MRC-5 cells were mock-infected or infected with HCoV-OC43 (F) or HCoV-229E (G) at an MOI of 0.5 TCID 50 /cell and treated with NTZ (1 μg/mL) or vehicle immediately after the virus adsorption period. After 24 h proteins were digested with Endo-H (+) or left untreated (−) and processed for IB analysis using anti-S HCoV-OC43 (F) or anti-S HCoV-229E (G) antibodies, and quantitated by scanning densitometry. The Endo-H-cleaved faster-migrating S forms are indicated by white arrowheads. The percentage of Endo-H-resistant (Endo-H R) S protein in the different samples is indicated.

Article Snippet: Nitazoxanide [2-acetyloxy- N -(5-nitro-2-thiazolyl) benzamide, Alinia] (NTZ) and tizoxanide (TIZ; Romark Laboratories, L.C.), remdesivir (MedChemExpress), ribavirin and chloroquine (Sigma-Aldrich), dissolved, respectively, in DMSO stock solution (NTZ, TIZ, remdesivir) or water (ribavirin, chloroquine), were diluted in culture medium, added to infected cells after the virus adsorption period, and maintained in the medium for the duration of the experiment, unless differently specified.

Techniques: Infection, Virus, Adsorption, Molecular Weight