Journal: Nature chemical biology

Article Title: A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation

doi: 10.1038/nchembio.2536

Figure Lengend Snippet: (a) Scheme depicting theoretical possibilities for H2O2-derived oxidizing equivalents to reach and oxidize redox-regulated proteins. Left column, canonical flow of oxidizing equivalents from H2O2 to NADPH through Prx1/2, Trx1 and TrxR1. Middle column, branch points (i–iv) potentially involved in the delivery of oxidizing equivalents to redox-regulated target proteins. Right column, reduction of oxidized target proteins by the thioredoxin system. All molecules are shown in the oxidized state. (b) Scheme depicting the mechanism-based kinetic trapping approach used to monitor cytosolic protein thiol oxidation. Prior to cell lysis, free thiols are blocked with N-ethylmaleimide (NEM; black squares). Cells are osmotically disrupted to release cytosolic proteins. Disulfide-containing proteins are selectively captured by the immobilized recombinant trapping mutant of human Trx1. (c,d) HAP1 cells proficient (wild type, WT) or deficient (ΔPrx1+2) in Prx1+2 expression (c) and HEK293T cells induced to express either scrambled (SCR) or specific (Prx1+2) shRNA (d) were exposed to the indicated concentrations of H2O2 for 15 s. Overall protein thiol oxidation, reflected by the formation of Trx-S-S-X conjugates, was assessed by the kinetic trapping approach and analyzed by immunoblotting against the SBP tag of the Trx1 trapping mutant. In c and d, different types of Trx conjugates are indicated. The Trx-S-S-Prx and Trx-S-S-(Prx)2 conjugates seen in cells lacking Prx1+2 represent trapping of other members of the Prx family (see Supplementary Fig. 3). Uncropped blots for c and d are shown in Supplementary Figure 4. IB, immunoblotting; NR, nonreducing; R, reducing conditions; SBP, streptavidin-binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥3 independent experiments.

Article Snippet: Open reading frames and the streptavidin binding peptide (SBP) tag were amplified by PCR and ligated into pcDNA3.1(−) using the Gibson Assembly Cloning Kit (New England BioLabs).

Techniques: Derivative Assay, Lysis, Recombinant, Mutagenesis, Expressing, shRNA, Western Blot, Binding Assay

Journal: Nature chemical biology

Article Title: A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation

doi: 10.1038/nchembio.2536

Figure Lengend Snippet: (a,b) HAP1 cells proficient (WT) or deficient (ΔPrx1+2) in Prx1+2 expression (a) and HEK293T cells induced to express scrambled (SCR) or specific (Prx1+2) shRNA (b) were treated for 1 h with 10 μM (a) or 20 μM (b) auranofin or solvent control (DMSO), or were left untreated (UT). Overall protein thiol oxidation was assessed by kinetic trapping and analyzed by immunoblotting (IB). (c) HEK293T cells induced to express scrambled or specific (Prx1+2) shRNA were transfected with scrambled or specific (Trx1) siRNA and exposed to the indicated concentrations of H2O2 for 15 s. Overall protein thiol oxidation was assessed by kinetic trapping and analyzed by immunoblotting against the SBP tag of the Trx1 trapping mutant. Uncropped blots are shown in Supplementary Figure 7. NR, nonreducing; R, reducing conditions; SBP, streptavidin binding peptide; Trx-Trap, eluate from Trx trapping beads. Blots are representative of ≥ 3 independent experiments.

Article Snippet: Open reading frames and the streptavidin binding peptide (SBP) tag were amplified by PCR and ligated into pcDNA3.1(−) using the Gibson Assembly Cloning Kit (New England BioLabs).

Techniques: Expressing, shRNA, Western Blot, Transfection, Mutagenesis, Binding Assay

Journal: Nature chemical biology

Article Title: A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation

doi: 10.1038/nchembio.2536

Figure Lengend Snippet: (a) HEK293T cells expressing SBP-tagged Prx family members (1, 2, 3, 5 and 6) were exposed to 100 μM H2O2 for 1 min (+) or left untreated (−). Free thiols were blocked by NEM before cell lysis. Mixed disulfide intermediates (outlined by dashed-line rectangles) were visualized on nonreducing gels by immunoblotting against the streptavidin binding peptide (SBP) tag. The blot is representative of three independent experiments. EV, empty vector. (b) Scheme explaining the pattern of proteins on two-dimensional nonreducing/reducing diagonal gels. *, interaction partners (X) released from X-S-S-Prx conjugates; **, interaction partners (X) released from X-S-S-(Prx)2 conjugates; ***, Prx-SBP or Prx (end.) released from X-S-S-Prx or X-S-S-(Prx)2 conjugates; #, Prx-SBP or Prx (end.) that was originally in the monomeric form; ##, Prx-SBP or Prx (end.) that was originally in the dimeric form; end., endogenous. Red and blue marks indicate molecular weight markers. (c,d) 2 × 108 HEK293T cells expressing either wild-type (WT), resolving cysteine-deficient (ΔCR) or double (resolving and peroxidatic) cysteine-deficient (ΔCPΔCR) Prx1-SBP (c) or Prx2-SBP (d) were exposed to 100 μM H2O2 for 3 min. Following thiol blocking and affinity purification, covalent interactions were analyzed by two-dimensional nonreducing/reducing diagonal SDS–PAGE. In c, the cartoons illustrate the nature of the disulfide linked complexes that lead to the formation of the two lower diagonals. In c and d, the arrows indicate the direction of protein migration.

Article Snippet: Open reading frames and the streptavidin binding peptide (SBP) tag were amplified by PCR and ligated into pcDNA3.1(−) using the Gibson Assembly Cloning Kit (New England BioLabs).

Techniques: Expressing, Lysis, Western Blot, Binding Assay, Plasmid Preparation, Molecular Weight, Blocking Assay, Affinity Purification, SDS Page, Migration

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Infection of HepaRG with HEV-3f. (A) HEV RNA (GE/ml supernatant) in the supernatant of HepaRG cells infected at MOI 100 (GE/cells) with HEV-3f for up to 100 days. Means from triplicate samples ± SD are shown. (B) Detection of HEV capsid (ORF2) in the lysates of HepaRG cells infected with HEV-3 at a MOI of 100 (GE/cells) at several days post-infection (d.p.i.). (C) Detection of HEV ORF2 by fluorescence microscopy after staining with an anti-ORF2 antibody in HepaRG cells not-infected (NI) or infected with HEV-3f at MOI 100 GE/cells for 100 days. Nuclei were stained using DAPI. Scale bars: 10 μM.

Article Snippet: The membrane was then incubated with the required dilution of specific antibodies raised against ORF2 (mouse, 1/2000 dilution, clone 1E6, Merck Millipore), actin (mouse, 1/2000 dilution, clone M2, Sigma), DDX58 (mouse, 1/1000 dilution, clone Alme-1, AdipoGen Life Sciences), IFIH1 (rabbit, 1/1000 dilution, AT113, ALX-210-935, Enzo Life Sciences), or IRF1 (rabbit, 1/1000 dilution, VPA00801, Bio-Rad).

Techniques: Infection, Fluorescence, Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: Identification of interferon-stimulated genes with modulated expression during hepatitis E virus infection in pig liver tissues and human HepaRG cells

doi: 10.3389/fimmu.2023.1291186

Figure Lengend Snippet: Validation of the PCR array data. (A) HepaRG cells were infected or not with HEV-3f for 100 days at MOI 100 and the expression of selected genes shown by PCR array to be up-regulated or unchanged was analyzed by RT-qPCR. The results shown are the geometric means ± SD from 3 to 4 replicate samples and are representative of 2 independent experiments. GUSB , B2M and GAPDH were used as reference genes. Unpaired t-test with Welch’s correction Mann-Whitney test, *: p<0.05 (B) Immunoblot showing the expression of the protein encoded by IFIH1 , DDX58 and IRF1 in HepaRG cells infected with HEV-3f or not (NI) for 100 days at MOI 100 for 3 replicate samples. Representative blots from 2 independent experiments are shown. ORF2 and actin protein levels were also detected as control of infection and loading, respectively. (C) Detection of CXCL10 in the supernatant of non-infected HepaRG (NI) or HepaRG cells infected with HEV-3f for 100 days at MOI 100 (GE/cell) by ELISA. The results shown are the means ± SD from 4 replicate samples and are representative of 2 independent experiments. Unpaired t-test with Welch’s correction, *: p<0.05.

Article Snippet: The membrane was then incubated with the required dilution of specific antibodies raised against ORF2 (mouse, 1/2000 dilution, clone 1E6, Merck Millipore), actin (mouse, 1/2000 dilution, clone M2, Sigma), DDX58 (mouse, 1/1000 dilution, clone Alme-1, AdipoGen Life Sciences), IFIH1 (rabbit, 1/1000 dilution, AT113, ALX-210-935, Enzo Life Sciences), or IRF1 (rabbit, 1/1000 dilution, VPA00801, Bio-Rad).

Techniques: Biomarker Discovery, Infection, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Control, Enzyme-linked Immunosorbent Assay