Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: Role of the nuclear transcription factor RARB in altering the expression of genes involved in steroidogenesis. ( A ) H295R cells were transfected with the human −1.05 kb HSD3B2, −1.3 kb StAR and −3.7 kb CYP17A1, -2327 bp CYP11A1 promoter constructs along with an expression vector for the RARB transcription factor. Transfection medium was changed after 6 h and cells were cultivated in serum free medium for 24 h. Thereafter, cells were grown in the presence or absence of ATRA in serum-free medium for another 24 h. Promoter activation was assessed by the dual luciferase assay (Promega) using pRL-TK as internal control. Data are expressed in Relative Light Units (RLU). Error bars show the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01. ( B ) Effect of RARB overexpression in absence or presence of ATRA was studied with respect to endogenous StAR and HSD3B2 expression. H295R cells were transiently transfected with RARB or the empty vector for control. Transfected cells were optionally treated with ATRA for 24 h in starvation medium. Finally, the effect of RARB on StAR and HSD3B2 was assessed by performing specific Western blot analyses. A representative blot is shown on the left and quantitative analysis on the right. Data are the mean ± SD of three independent experiments. ( C ) Effect of RARB overexpression and ATRA treatment on H295R steroid production. H295R cells were transiently transfected with RARB or the empty vector for control. Transfected cells were optionally treated with 1 μM ATRA for 24 h in starvation medium. Steroid production was labeled with [ 3 H] pregnenolone for 90 min and extracted steroids were resolved by TLC. A representative TLC is shown on the left and the quantitative analysis of steroids shown on the right. Data are the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01. Prog, progesterone; Δ4A, androstenedione; Preg, pregnenolone; 17OH Preg, 17α-hydroxypregnenolone; 17OH Prog, 17OHP; 11DOC, 11-deoxycortisol.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Activation Assay, Luciferase, Control, Over Expression, Western Blot, Labeling

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: ANGPTL1 suppresses CYP17A1 expression through inducing ERK1/2 phosphorylation in starved H295R cells. ( A ) Effect of rANGPTL1 protein on ERK and AKT kinase phosphorylation: Western blot analysis was performed on starved H295R cells treated with 50 ng/ml rANGPTL1 for 0-4 h to study the changes in phosphorylation of ERK1/2 and AKT kinases. Western blot was performed on total protein preparations from treated starved H295R cells. Levels of total ERK and AKT were also analyzed. ( B ) Effect of rANGPTL1 treatment on steroidogenic CYP17A1 expression. Western blot analysis showed CYP17A1 expression was suppressed under treatment with rANGPTL1 in 30 min. We used β-actin as loading control. ( C ) The effect of inhibiting MEK/ERK1/2 phosphorylation on CYP17A1 expression in ANGPTL1 treated starved H295R cells. Cells were starved for 48 hrs and treated or not with rANGPTL1. When indicated, cells were pretreated with 10 μM U0126 for 30 min before rANGPTL1 treatment. Cells were lysed and levels of CYP17A1 and pERK1/2 were analyzed by Western blotting. Levels of total ERK were taken as a loading control. ( D ) Effect of rANGPTL1 on DUSP6 expression. Levels of DUSP6 were assessed by Western blotting. Representative blots are shown on the left and the quantitative analysis of blots is shown on the right. Data are the mean ± SEM of two or three independent experiments. * p < 0.05, ** p < 0.01.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Expressing, Phospho-proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

doi: 10.1038/srep10132

Figure Lengend Snippet: Scheme of suggested regulation of androgen production by ANGPTL1 in H295R cells based on presented findings (arrows) and information from literature . ANGPTL1 enhances ERK1/2 phosphorylation and thereby modulates CYP17A1 expression. ANGPTL1 also stimulates DUSP6 expression. DUSP6 and ERK1/2 regulate each other; while DUSP6 may dephosphorylate ERK1/2, phospho-ERKs phosphorylate DUSP6 prompting its degradation.

Article Snippet: Rabbit polyclonal antibody against human CYP17A1 was custom made by Genscript (Piscataway, NJ, USA) .

Techniques: Phospho-proteomics, Expressing

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: Recombinant L. major expressing GM-CSF as part of an episomal vector. (A) Expression vectors pNEO-mGM-CSF and pNEO-hGM-CSF were made by inserting the murine mGM-CSF and hGM-CSF genes, respectively, downstream of the neomycin phosphotransferase (NEO) expression cassette as described in Materials and Methods. Arrows indicate the orientation of GM-CSF transcript maturation. In pNEO-hGM-CSF/R, the hGM-CSF gene is cloned in the opposite orientation relative to mRNA maturation signals. Bg, BglII. (B) Southern blot of L. major genomic DNA digested with BglII and hybridized to the murine and human GM-CSF-specific probes. Lanes: 1, L. major pNEO control; 2, L. major expressing either the mGM-CSF or the hGM-CSF gene. (C) GM-CSF expression in L. major GM-CSF recombinant cells. A Northern blot of total Leishmania RNA hybridized to the same probes as above. Lanes 1 and 2 are as in panel B. Lane 3, L. Major expressing the hGM-CSF gene.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Recombinant, Expressing, Plasmid Preparation, Clone Assay, Southern Blot, Northern Blot

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: Stable integration of the GM-CSF gene into the L. major LV39 genome. (A) Schematic representation of the L. major rRNA locus with the RNA pol I promoter indicated by a flag. The GM-CSF expression cassette was inserted downstream of the rRNA promoter for stable and high expression. The EcoRV (EV) and BamHI (B) restriction sites and the size of the restriction fragments are indicated. (B) Southern blot hybridization of the L. major LV39 wild-type strain (lane 1) and of L. major mGM-CSF recombinant strain made by the integration of the mGM-CSF gene into the ribosomal locus (lane 2) with probes specific to the rRNA promoter sequence and the mGM-CSF gene, respectively. The EcoRV restriction fragments confirming the correct integration of the GM-CSF gene into the parasite genome are indicated by arrows. (C) Clamped homogeneous electric field electrophoresis and Southern blot hybridization of L. major chromosomes (lanes and probes are as indicated in panel B). The ribosomal locus is part of chromosome 27. (D) Northern blot hybridization of L. major total RNA (lanes are as indicated in panel B) with the GM-CSF gene probe. RNA loading was monitored by ethidium bromide staining (lower panel).

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Expressing, Southern Blot, Hybridization, Recombinant, Sequencing, Electrophoresis, Northern Blot, Staining

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: GM-CSF release by intracellular Leishmania expressing the GM-CSF gene. (A) CM-CSF production and release within macrophages at different time points following infection of J774 macrophages with L. major expressing the episomal form of the mGM-CSF gene (□) or with L. major wild type (•). The amount of GM-CSF in lyzed macrophages was measured by ELISA. (B) CM-CSF release in the extracellular milieu of macrophages infected with L. major expressing the episomal form of the mGM-CSF gene (▴) or with L. major wild type (○). The amount of GM-CSF in the culture supernatants of infected macrophages was measured by ELISA. Dilutions of both the capture and biotinylated antibodies were at 1/250.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Expressing, Infection, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: GM-CSF detection by immunofluorescence within macrophages infected with GM-CSF-producing parasites. After overnight infection of J774 murine macrophages with either L. major LV39 expressing the integrated mGM-CSF gene or L. major expressing the episomal GM-CSF vector, macrophages were washed, fixed, and then incubated with the monoclonal anti-mouse GM-CSF antibody and Alexa Fluor 546, as indicated in Materials and Methods. Coverslips were then mounted and sealed on the microscope slide for confocal observation. Color images were created with Fluoview 300 version 3.3 software, and color contrasts were adjusted using Photoshop version 6.0 software. (A) macrophages infected with L. major wild type (dilutions are at 1/250 for the first antibody and 1/1,000 for the second antibody), (B) macrophages infected with L. major expressing the episomal form of the mGM-CSF gene (dilutions are at 1/250 for the first antibody and 1/1,000 for the second antibody), (C) uninfected macrophages (dilutions are at 1/1,000 for the first antibody and 1/2,000 for the second antibody), and (D) macrophages infected with L. major LV39 expressing the integrated form of the mGM-CSF gene (dilutions are at 1/1,000 for the first antibody and 1/2,000 for the second antibody).

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Immunofluorescence, Infection, Expressing, Plasmid Preparation, Incubation, Microscopy, Software

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: Intracellular killing of L. major amastigotes expressing the mGM-CSF and/or the hGM-CSF gene. J774 murine macrophages and/or human monocytes differentiated to macrophages (5 × 104 cells/well) were incubated for 6, 48, or 72 h with late-stationary-phase L. major promastigotes (parasite-to-cell ratio, 20:1) expressing either the mGM-CSF or the hGM-CSF gene as described in Materials and Methods. At these fixed time points, cell cultures were dried and stained with Diff Quik to determine the level of infection. (A) Infection of murine macrophages with the L. major pNEO control, L. major mGM-CSF expressing the episomal mGM-CSF gene, and L. major mGM-CSF incubated with an anti-GM-CSF neutralizing antibody prior to macrophage infection. (B) Infection of human monocytes with L. major pNEO, L. major hGM-CSF, and L. major hGM-CSF/R (with the human GM-CSF gene cloned in the reverse orientation). The results shown here are the mean of three independent experiments with duplicate samples and the standard errors of the mean.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Expressing, Incubation, Staining, Diff-Quik, Infection, Clone Assay

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: Outcome of cutaneous disease in susceptible BALB/c mice infected with late-stationary-phase L. major promastigotes expressing the murine GM-CSF gene. The infection was monitored for up to 7 weeks postinoculation as described in Materials and Methods. Results obtained with mice infected with the L. major NEO control, mice infected with the L. major LV39 recombinant strain expressing the mGM-CSF gene integrated into the 18S ribosomal locus under the control of the rRNA promoter, and mice infected with L. major GM-CSF expressing the episomal form of the mGM-CSF gene are shown. The result shown here is the average mean of three independent experiments.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Infection, Expressing, Recombinant

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: A time course pattern of cytokine expression by J774 macrophages infected with L. major LV39 expressing the integrated mGM-CSF gene. Graphs represent densitometric quantification of bands from cytokine mRNA (IL-1Ra, IL-1β, IL-18, and IL-6) normalized to the GAPDH signal. LPS was used as a control. The relative increase in the levels of the various cytokines is in comparison to macrophages infected with the wild-type parasites. The data presented here are the mean of three independent experiments. Error bars, ± 0.1 to 0.5.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Expressing, Infection

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: Patterns of chemokine expression by J774 macrophages infected with L. major LV39 expressing the integrated mGM-CSF gene. Graphs represent densitometric quantification of bands from chemokine mRNA (RANTES, MCP-1, MIP-2, MIP-1α, and MIP-1β) normalized to the GAPDH signal. The relative increase in the levels of the various chemokines is in comparison to macrophages infected with the wild-type parasites is shown. The data presented here are the mean of three independent experiments. Error bars, ± 0.03 to 0.4.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Expressing, Infection

Journal:

Article Title: Recombinant Leishmania major Secreting Biologically Active Granulocyte-Macrophage Colony-Stimulating Factor Survives Poorly in Macrophages In Vitro and Delays Disease Development in Mice

doi: 10.1128/IAI.71.11.6499-6509.2003

Figure Lengend Snippet: No inhibition in mice infected with GM-CSF-expressing recombinant parasites. BALB/c mice infected either with L. major LV39 expressing the integrated mGM-CSF gene (L.m-GM-CSF) or with the L. major NEO strain (L.m-NEO) were treated with aminoguanidine, a selective inhibitor of inducible iNOS synthesis, and the outcome of the cutaneous infection in BALB/c mice was monitored for up to 4 weeks and compared to that for the untreated animals. The data presented here are the mean of two independent experiments.

Article Snippet: The capture monoclonal anti-mouse GM-CSF antibody (51-26201E; BD Biosciences) was used in combination with the biotinylated mGM-CSF affinity-purified polyclonal detection antibody (51-26202E; BD Biosciences) in a mouse GM-CSF ELISA.

Techniques: Inhibition, Infection, Expressing, Recombinant

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Viral titer of rVSVs expressing G, ANDV Gn/Gc, or SNV Gn/Gc in human or mouse primary lung endothelial cells. Means ± SD: n = 6 wells of infected cells examined over three independent experiments. b Surface expression of endogenous PCDH1 in primary mouse lung microvascular endothelial cells (MLMECs) used in ( a ). Cells were immunostained with PCDH1-specific monoclonal antibody (mAb) 3305 or a negative control mAb (Ctrl.) Scale bar, 20 µm. c Infectivity of rVSVs bearing HTNV, ANDV, or SNV Gn/Gc in primary MLMECs expressing flag-tagged, wild-type (WT) or mouse-variant (F83L) human PCDH1. rVSV infectivities are expressed as fold change relative to that in non-complemented cells (set to one). Means ± SD: n = 35 wells of infected cells examined over three independent experiments. d Cells described in ( c ) were immunostained with an anti-flag antibody to detect total human PCDH1 expression in transfected MLMECs. Scale bar, 20 µm. e Alignment of human PCDH1-EC1 amino acid sequences with a selection of rodent and primate species. All of the residues within EC1 which deviate from the consensus are shown and highlighted. Residues that specifically deviate from human EC1 are indicated [green, experimentally tested SNV-susceptible hosts; purple, experimentally unknown; orange, residues in EC1 that are different between human and other species with no known link to susceptibility]. Alignments generated by Clustal Omega. f The capacity of U2OS PCDH1 -KO cells expressing the indicated PCDH1 variants to support hantavirus Gn/Gc-dependent entry. Cells were exposed to rVSVs bearing the indicated Gn/Gc proteins or authentic ANDV, SNV, or HTNV. “None” indicates no PCDH1 expression. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. rVSV means ± SD: n = 8 wells of infected cells examined over three independent experiments. ANDV/SNV/HTNV means ± SD: one experiment examining n = 3 (ANDV and SNV) or n = 10 (HTNV) wells of infected cells. Infectious units ( a ) were compared by unpaired, two-tailed t test with Welch’s correction. Infectivities ( c , f ) were compared by one-way ANOVA with Dunnett’s correction for multiple comparisons; ns > 0.05, ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Expressing, Infection, Negative Control, Variant Assay, Transfection, Selection, Generated, Virus, Two Tailed Test

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Diagram of direct binding ELISA comparing sEC1-2(WT) and sEC1-2(F83L) capture of rVSVs. b Direct binding ELISA. rVSVs expressing ANDV, SNV, or HTNV Gn/Gc were added to sEC1-2(WT) or sEC1-2(F83L) coated ELISA plates. Means ± SD: n = 4 wells examined over two independent experiments. A, absorbance. c Diagram depicting competition ELISA comparing sEC1-2(WT) and sEC1-2(F83L) as competitive reagents. d Competition ELISA. rVSVs expressing ANDV or SNV Gn/Gc were pre-incubated with sEC1-2(WT) or sEC1-2(F83L) before added to sEC1-2(WT) coated ELISA plates. The ELISA signal was normalized to that obtained without competing sEC1-2. Means ± SEM: n = 7 wells examined over three independent experiments (ANDV), n = 4 wells examined over two independent experiments (SNV). e Diagram depicting infection-inhibition assay comparing sEC1-2(WT) and sEC1-2(F83L) as inhibiting reagents. f Infection-inhibition assay using sEC1-2(WT) and sEC1-2(F83L) to block infection (MOI of 0.1) of rVSVs bearing ANDV or SNV Gn/Gc on primary human endothelial cells (HUVECs). The infectivity of each virus was normalized to that obtained without sEC1-2. Averages ± SD: n = 6 wells examined over two independent experiments. (sEC1-2, soluble extracellular cadherin domains 1 and 2). Figures ( a , c , e ) were created with BioRender.com. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, Incubation, Infection, Inhibition, Blocking Assay, Virus

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Relative infectivity of rVSVs bearing ANDV, SNV, or HTNV Gn/Gc on U2OS PCDH1- KO cells complemented with WT or mutant PCDH1. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 wells of infected cells examined over three independent experiments (rVSV-ANDV-Gn/Gc infection on U2OS PCDH1- KO cells complemented with V86A had n = 8). Infectivities were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. b Relative infectivity of authentic ANDV, SNV, or HTNV on the cell lines described in ( a ). The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 infected wells were examined over three independent experiments (HTNV infection on U2OS PCDH1- KO cells complemented with D85A and F83A/D85A had n = 6 wells of infected cells examined over two independent experiments). For ANDV and SNV infection on the control cell line, U2OS PCDH1- KO cells complemented with WT, n = 12 (SNV) and n = 18 (ANDV) wells of infected cells were examined over four independent experiments. Infectivities were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Infection, Mutagenesis, Virus, Control

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Crystal structure of the proposed anti-parallel EC1-4 trans-dimer. Structure is in the “open conformation” displaying residues representing three degrees of binding strength to ANDV Gn/Gc. Residues that when mutated bind similarly to WT (green); those that display a mild reduction in binding (orange); and those that display a strong reduction in binding (purple). The Gn/Gc binding site relative to the EC1:EC4 adhesive interface (dark blue) is indicated. An alternative view of the EC1:EC4 binding interface is shown to the right. Structure adapted from PDB 6MGA. b Competition ELISA using WT and mutant sEC1-2 as competitive reagents to the binding of rVSV-ANDV-Gn/Gc to WT sEC1-2 coated wells. Averages ± SD: n = 4 wells of each sEC1-2 dilution examined over two independent experiments [ n = 3 for one of the dilutions of sEC1-2(E137R)]. c Relative infectivity of rVSVs bearing ANDV, SNV, or HTNV Gn/Gc on U2OS PCDH1- KO cells complemented with WT or ΔEC4 PCDH1. The infectivity of each virus was normalized to that obtained in U2OS PCDH1 -KO cells complemented with WT PCDH1. Means ± SD: n = 9 wells of infected cells examined over three experiments. d Schematic representation of WT or mutant sEC1-4 proteins forming monomers or dimers. e Non-reduced and reduced purified WT and mutant sEC1-4 were separated on an SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue staining. kDa, kilodalton. f Non-reduced samples in ( e ) were run on a native-polyacrylamide gel and visualized as in ( e ). A representative gel from one experiment of two independent experiments is shown for ( e ) and ( f ). g ELISA detecting WT and mutant sEC1-4 coated plates, using an anti-Flag-HRP antibody. Mean ± SD: n = 4 wells examined over two independent experiments. ( h ) Capacity of rVSV-ANDV-Gn/Gc to bind to WT or mutant sEC1-4 coated plates. Done in parallel with ( g ). Mean ± SD: n = 4 wells of each viral particle dilution examined over two independent experiments. Infectivities ( c ) and ELISA signal ( g ) were compared by one-way ANOVA with Dunnett’s test for multiple comparisons. (sEC1-4, soluble extracellular cadherin domains 1–4). Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Binding Assay, Adhesive, Enzyme-linked Immunosorbent Assay, Mutagenesis, Infection, Virus, Purification, Staining

Journal: Nature Communications

Article Title: Two point mutations in protocadherin-1 disrupt hantavirus recognition and afford protection against lethal infection

doi: 10.1038/s41467-023-40126-y

Figure Lengend Snippet: a Reference nucleotide and amino acid sequence of PCDH1-EC1 Syrian hamster (WT, above) and representative sequences and trace files of Syrian hamsters after CRISPR-Cas9 genome editing [PCDH1(F83A/D85R), lower left] and [PCDH1(10a.a.) lower right]. The nucleotides encoding the corresponding human PCDH1-EC1 Gn/Gc-interacting residues, are highlighted: F83 in purple and D85 in green along with the location of the single guide RNAs (KI, knock-in; sgRNA, single guide RNA). b Immunoblot detecting PCDH1 in lung tissue lysates from WT or CRISPR knock-in mutant Syrian hamsters. Antibody targets PCDH1’s cytoplasmic tail. kDa, kilodalton. A representative blot from a single experiment of two independent experiments is shown. Uncropped blots in Source Data. c Syrian hamster ANDV challenge. Groups of WT, PCDH1(F83A/D85R), and PCDH1(10a.a.) CRISPR knock-in mutant hamsters were inoculated intranasally with ANDV (2,000 PFU). Mortality was monitored and hamsters were euthanized on day 35 post-exposure. One experiment was performed, with n = 8 hamsters for each group. Data was analyzed using two-sided, log-rank Mantel–Cox test. d Lung sections from WT and PCDH1(F83A/D85R) hamsters were collected 15 days post ANDV exposure. Representative histochemical images indicate inflammation in pulmonary tissue (left), ANDV nucleoprotein (N) (middle, tan staining), and ANDV RNA (right, red staining, detected by in situ hybridization). Representative images from one experiment from one out of three hamsters from each group are shown. Scale bars represent 100 µm. Figure ( a ) includes an image from Flaticon.com. Source data are provided as a Source Data file.

Article Snippet: Briefly, U2OS PCDH1 -KO cells, complemented with WT or mutant PCDH1, were exposed to virus at an MOI of 0.5 (ANDV), 1.5 (SNV), or 3 (HTNV) plaque-forming units (PFU) per cell, and viral infectivity was determined by immunostaining of formalin-fixed cells at 72 h post-infection using a 1:5,000 dilution of rabbit polyclonal antibodies (pAbs) detecting ANDV (NR-9673, BEI Resources), HTNV (NR-12152, BEI Resources) or SNV (NR-9674, BEI Resources) nucleoproteins.

Techniques: Sequencing, CRISPR, Knock-In, Western Blot, Mutagenesis, Staining, In Situ Hybridization