pxr003 casrx crrna  (Addgene inc)

Journal: iScience

Article Title: Cell surface heparan sulfate is an attachment receptor for grass carp reovirus

doi: 10.1016/j.isci.2025.112033

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Article Snippet: pXR003 , Konermann et al. , Addgene Plasmid # 109053.

Techniques: Virus, Recombinant, Saline, Purification, Adjuvant, Control, Lysis, Membrane, Cloning, cDNA Synthesis, CCK-8 Assay, Plasmid Preparation, Software

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A ) Immunohistochemical staining of lung biopsies of controls and COVID-19 patients, using anti-FABP4 antibody. ( B , C ) Circulating FABP4 concentrations measured from ( B ) female ( n = 109 female patients) and ( C ) male COVID-19 patients ( n = 174 male patients) and healthy controls ( n = 32 female and n = 13 male), stratified based on disease severity (**** p < 0.0001, ** p = 0.002, * p = 0.0221). ( D ) Circulating IL-6 levels in COVID-19 patients, stratified based on disease severity (** p = 0.0017). ( B – D ) Statistical analysis was performed using one-way ANOVA. ( E , F ) Effect size estimates and inference based on regression analysis of FABP4 concentration on ( E ) COVID-19 severity and ( F ) oxygen support measures while accounting for time of collection post symptom onset, age, sex, and BMI, using the linear mixed model to account for the patient-level random effects. ( E ) The healthy controls and, ( F ) patients who did not require oxygen support were used as a reference group. p-values are calculated based on the Wald test. For ( B – F ), the analysis included n = 283 total patients and n = 45 healthy controls. ( G , H ) FABP4 concentration in patients with severe and critical disease ( n = 176 patients), stratified based on ( G ) presence or absence of comorbidities (listed in Table and Dataset , *** p = 0.0009) or ( H ) age (*** p = 0.0006). Statistical analysis was performed using Welche’s t-test. ( B – H ) Data are derived from patient cohort 1 (collected November 2020–May 2021). Patients were sampled longitudinally, and the data shown in ( B ), ( C ), ( D ), ( G ) and ( H ) represent the maximum measured concentration per patient. Data are shown as the mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Immunohistochemical staining, Staining, Concentration Assay, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A , B ) Maximum concentrations of circulating ( A ) FABP4 (**** p < 0.0001, * p = 0.044) and ( B ) IL-6 (*** p = 0.0005) of cohort 2 of COVID-19 patients ( n = 166) stratified based on disease severity (moderate: n = 52, severe: n = 21, and critical: n = 42). ( C ) Circulating levels of C-reactive protein (**** p < 0.0001, *** p = 0.0002, * p = 0.0221), ( D ) leukocytes (**** p < 0.0001, ** p = 0.0015) and ( E ) lymphocytes (**** p < 0.0001) of COVID-19 patients measured on the day in which the maximum FABP4 concentration was measured (day post symptom onset). Statistical analysis was performed using one-way ANOVA ( n = 283 cohort 1, and n = 116 cohort 2). ( F – I ) Maximum FABP4 concentration pooled from severe and critically ill patients (cohort 1: n = 176, cohort 2: n = 63), stratified based on ( F ) the presence or absence of comorbidities (listed in Table and Dataset , **** p < 0.0001), ( G ) age (** p = 0.0011), ( H ) BMI (*** p = 0.0001, ** p = 0.0098 and 0.004, * p 0.0263), and ( I ) the presence or absence of cardiometabolic conditions (diabetes, hypertension, or coronary artery disease, **** p < 0.0001). Statistical analysis for ( F ), ( G ) and ( I ) were performed using Welch’s t-test and one-way ANOVA for ( H ). Data are shown as the mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – C ) hTERT pre-adipocytes and differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 1). ( A , B ) Relative expression of viral ( A ) genomic RNA (nucleocapsid, *** p = 0.0002, ** p = 0.0018) and ( B ) sub-genomic RNA (ORF1ab, *** p = 0.0003, * p = 0.0263), normalized to β-actin. ( C ) Viral loads measured from supernatant using plaque assay. (**** p < 0.0001, ** p = 0.0024). Data are pooled from two independent experiments ( n = 8, biological replicates). Statistical analysis was performed using two-way ANOVA. ( D ) IL-6 levels in the supernatant of differentiated adipocytes with or without viral infection (MOI = 1), measured by ELISA. Data are pooled from three independent experiments ( n = 14, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( E – G ) Adipocytes were infected at 4, 9, 12, 16, and 20 days post-differentiation (MOI = 1), with measurements taken 48 h post-infection. ( E ) Viral loads measured by plaque assay; data represent two independent experiments ( n = 4, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( F ) Western blot of viral nucleocapsid, FABP4, and β-actin proteins levels in cell lysates. ( G ) Quantification of nucleocapsid and FABP4 band intensities normalized to β-actin, representative of two independent experiments ( n = 4, biological replicates, **** p < 0.0001, *** p = 0.0001 and 0.0006). Statistical analysis was performed using two-way ANOVA. ( H , I ) Percent area of lipid droplets in infected cells and neighboring cells, quantified by fluorescence neutral lipid staining (Bodipy). Infected cells identified by ( H ) nucleocapsid-positive staining and ( I ) dsRNA-positive staining. Data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001, *** p = 0.0001); statistical analysis was performed using a standard t-test. ( J ) Representative confocal images of infected differentiated adipocytes (MOI = 1), stained for dsRNA (red), lipid droplets (yellow), and nucleus (DAPI, blue). Scale bar = 50 μm; magnified regions = 10 μm ( n = 3). ( K ) Percent lipid droplet area relative to dsRNA-positive area per cell. Pearson correlation coefficient indicated as r. ( L , M ) Representative confocal images of control and infected adipocytes at 8, 12, 24, and 48 h post-infection, stained for dsRNA (red), lipid droplets (yellow), calnexin (blue), and FABP4 (gray). ( L ) Merged image of all stains (Scale bar = 50 μm); insets highlight overlap of FABP4, dsRNA, and calnexin (Scale bar = 5 μm). ( M ) Signal overlap between dsRNA, FABP4, and calnexin, and between FABP4 and calnexin individually (Scale bar = 5 μm). ( N – P ) Colocalization of target signals over time, represented as Pearson correlation R. Cells infected with MOI = 3 at 8 and 12 hpi; data pooled from two independent experiments ( n = 6, biological replicates, **** p < 0.0001). For 24 and 48 hpi, MOI = 1 was used ( n = 3) biological replicates. Statistical analysis was performed using one-way ANOVA. Data shown as mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Infection, Expressing, Plaque Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – D ) Pre-adipocytes and differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 0.1). ( A , B ) Relative expression of viral ( A ) genomic RNA (nucleocapsid, **** p < 0.0001, *** p = 0.0003 and 0.0004) and ( B ) sub-genomic RNA (ORF1ab, **** p < 0.0001), normalized to β-actin. ( C ) Viral loads measured from supernatant using plaque assay (**** p < 0.0001,*** p = 0.0001). Data are pooled from two independent experiments ( n = 8, biological replicates). Statistical analysis was performed using two-way ANOVA. ( D ) IL-6 measured by ELISA in the supernatant of differentiated adipocytes with or without virus infection (MOI = 0.1). Data are pooled from two independent experiments ( n = 8, biological replicates, **** p < 0.0001). Statistical analysis was performed using one-way ANOVA. ( E ) Western blots of SARS-CoV-2 nucleocapsid, FABP4, β-actin protein levels, and total protein (Ponceau S staining) in cell lysates of differentiated adipocytes infected with SARS-CoV-2 (MOI = 0.1 or MOI = 1). ( F ) Quantification of FABP4 band intensity normalized to total protein, representative of two independent experiments ( n = 4, biological replicates). ( G ) FABP4 gene expression relative to β-actin, pooled from two independent experiments ( n = 8, biological replicates). ( H ) FABP4 secretion in the supernatant within 1-hour incubation at the indicated time points following infection. Fold change is calculated relative to uninfected samples. Data are representative of two independent experiments ( n = 6, biological replicates, * p = 0.0336). Statistical analysis was performed using two-way ANOVA. ( I , J ) Representative confocal images of infected adipocytes stained with nucleocapsid (red), FABP4 (green), and lipid droplets (blue). ( I ) Low magnification and ( J ) high magnification images of the same samples (Scale bars = 50 μm, magnified regions = 10 μm) ( n = 3, biological replicates). ( K ) Percentage lipid droplet area relative to nucleocapsid-positive area per cell in infected differentiated adipocytes. Pearson correlation coefficient is indicated as r. Data are shown as the mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Infection, Expressing, Plaque Assay, Enzyme-linked Immunosorbent Assay, Virus, Western Blot, Staining, Gene Expression, Incubation

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A ) Percentage of FABP4 bound with fatty acid (BODIPY FL C12) in the presence or absence of CRE-14 or BMS309403 ( n = 3, technical replicates, **** p < 0.0001). ( B ) Representative MST time traces with blue and red regions indicating F cold and F hot , respectively, from which fluorescence measurements were normalized. ( C ) Dose-response curve showing FABP4 binding to increasing concentrations of CRE-14, represented as normalized fluorescence. KD value (954 nM) represents the average across two technical runs. ( D ) MRC5 cell viability following administration of titrated doses of CRE-14 at the indicated concentrations of FBS. ( E , F ) Differentiated adipocytes infected with SARS-CoV-2 (WA1/2020, MOI = 0.1) and treated with either CRE-14 (20 μM) or DMSO. ( E ) Relative RNA expression of nucleocapsid normalized to β-actin (**** p < 0.0001). ( F ) Viral load measured from the supernatant using plaque assay (**** p < 0.0001, *** p = 0.0004). ( G ) Representative confocal images of control and infected adipocytes (MOI = 1), fixed 48 h post-infection, stained for virus nucleocapsid (red), lipid droplets (yellow), and nuclei (DAPI, blue) ( n = 3, biological replicates). Scale bar = 500 μm. ( H ) Percentage of nucleocapsid-positive area per image, averaging 4–5 images per sample (**** p < 0.0001). ( I ) Representative confocal images showing lipid droplet content (yellow) in WT and FABP4-deficient adipocytes. ( J ) FABP4 -shRNA knockdown and scrambled controls infected with SARS-CoV-2 (WA1/2020, MOI = 1), with viral titers measured by plaque assay from supernatants. Data are pooled from two independent experiments (n = 8, biological replicates, **** p < 0.0001). Statistical analysis was performed using two-way ANOVA. ( K – M ) WT and FABP4-deficient differentiated adipocytes infected and treated with either DMSO, CRE-14, or BMS309403 at indicated doses, with cell lysates collected 48 h post-infection. Data are representative of two independent experiments ( n = 3). ( K ) Western blots showing nucleocapsid, FABP4, and total proteins (Ponceau S staining) in cell lysates. ( L , M ) Quantifications of nucleocapsid band intensity relative to total protein. Statistical analysis was performed using two-way ANOVA (**** p < 0.0001, * p = 0.017). ( N ) Percentage of lipid droplet area per cell in uninfected and SARS-CoV-2-infected adipocytes with or without FABP4 inhibitor treatment (20 μM, **** p < 0.0001, *** p = 0.0006). ( O , P ) Adipocytes infected at 5, 10, and 20 days post-differentiation (MOI = 1). ( O ) Western blot of nucleocapsid, FABP4, and GAPDH protein levels in cell lysates, and ( P ) viral titers in supernatant measured 48 h post-infection. Data are representative of two independent experiments ( n = 3, biological replicates, **** p < 0.0001, *** p = 0.0008, ** p = 0.0016). Statistical analysis was performed using two-way ANOVA. Data are shown as mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Fluorescence, Binding Assay, Infection, RNA Expression, Plaque Assay, Control, Staining, Virus, shRNA, Knockdown, Western Blot

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: ( A – E ) SARS-CoV-2-infected differentiated adipocytes (MOI = 1), treated with either DMSO, BMS309403 (20 μM) or CRE-14 (20 μM). ( A ) Relative RNA expression of SARS-CoV-2 nucleocapsid, normalized to β-actin. Data are pooled from two independent experiments ( n = 8, biological replicates, **** p < 0.0001, * p = 0.0229). ( B ) Quantification of nucleocapsid band intensity normalized to β-actin. Data are representative of three independent experiments ( n = 3, biological replicates, **** p < 0.0001, *** p = 0.0003, * p = 0.0182). ( C ) Western blot of nucleocapsid and β-actin protein levels in cell lysates. ( D ) Viral load measured by plaque assay, pooled from two independent experiments ( n = 8, biological replicates, **** p < 0.0001), representative of four independent experiments. ( E ) IL-6 levels in the supernatant of infected adipocytes treated with FABP4 inhibitors, measured by ELISA. Data are pooled from two independent experiments ( n = 8, biological replicates, *** p = 0.0004 and 0.0001). For ( A ), ( B ), ( D ), and ( E ), statistical analysis was performed using two-way ANOVA. ( F – H ) Infected wild-type (WT) and FABP4-deficient human adipocytes (MOI = 1). ( F ) Western blot of nucleocapsid, FABP4, and β-actin protein levels in cell lysates. ( G ) Quantification of nucleocapsid band intensity normalized to β-actin, representative of two independent experiments ( n = 3, biological replicates, ** p = 0.0015). ( H ) Viral load measured by plaque assay from supernatants collected 24 and 48 h post-infection. Data are pooled from three independent experiments ( n = 9, biological replicates, **** p < 0.0001). Statistical analysis was performed using a standard t-test. ( I – L ) Infected differentiated adipocytes (MOI = 3, biological replicates, **** p < 0.0001) fixed 48 h post-infection and stained with dsRNA (red), lipid droplets (yellow), and calnexin (blue). ( J , K ) Representative images of infected cells with ( J ) inhibitor treatment or ( K ) genetic deletion of FABP4. Scale bar = 50 μm, magnified regions = 10 μm. ( I , L ) Percentage dsRNA area and mean fluorescence intensity per cell. Statistical analysis was performed using one-way ANOVA for ( I ) and a standard t-test for ( L ) ( n = 3, biological replicates, **** p < 0.0001) biological replicates and 11 to 16 images per sample. ( M – O ) Real-time electric impedance traces and their corresponding median cell index (hours) for ( M ) and ( N ) wild-type and FABP4 knockout mouse pre-adipocytes infected with coronavirus OC43 at indicated MOIs. ( O ) MRC5 cells infected with OC43 and treated with either DMSO or CRE-14. Statistical analysis was performed using a standard t-test ( n = 3, biological replicates, **** p < 0.0001, ** p = 0.0026). Data are shown as mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Infection, RNA Expression, Western Blot, Plaque Assay, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Knock-Out

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: Syrian hamsters were infected intranasally with SARS-CoV-2 (Ank1, 100 TCID50) and treated daily with FABP4 inhibitor (CRE-14, 15 mg/kg) or vehicle. ( A ) Percent of initial body weight over time ( B ) Lung viral titers pooled from three independent experiments ( n = 18 for infected vehicle, n = 17 for CRE-14 treated, and n = 6 for each uninfected group). Statistical analysis was performed using two-way ANOVA for ( A ) (**** p < 0.0001) and a standard t-test for ( B ) (* p = 0.028). ( C , E ) Representative immunofluorescence and IHC staining of SARS-CoV-2 nucleocapsid in infected hamster lungs with or without CRE-14 treatment (Scale bar = 1 mm, n = 4). ( D ) Percentage of nucleocapsid-positive cells relative to total cell count, quantified from immunofluorescence staining ( n = 4, biological replicates, ** p = 0.0086). Statistical analysis was performed using a standard t-test. ( F ) Representative H&E staining of control and infected hamster lungs with or without inhibitor treatment (Scale bars: left = 2 mm, right = 200 μm). Low magnification images are shown in (Fig. ). ( G – L ) Pathology evaluation of lung histology in arbitrary units (A.U.), based on pathology scores ( n = 6 vehicle treated, n = 5 CRE-14 treated, further details in Table ). Statistical analysis was performed using a standard t-test (( G ) * p = 0.0391, ( H ) ** p = 0.0063, ( J ) * p = 0.0158, ( K ) * p = 0.014, ( L ) ** p = 0.002 and * p = 0.019). ( J ) Bronchial damage represents combined pathology scores of bronchial epithelial cell necrosis and presence of cellular debris in bronchi. ( L ) Alveolar damage represents combined pathology scores of alveolar epithelial cell necrosis, cellular debris in alveoli, hyaline membranes, fibrin deposition, and alveolar emphysema. ( M ) Representative Masson’s trichrome staining of control and infected hamster lungs with or without CRE-14 treatment (Scale bar = 1 mm, n = 4). Low magnification images are shown in (Fig. ). Data are shown as mean ± s.e.m. .

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Infection, Immunofluorescence, Immunohistochemistry, Cell Counting, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: FABP4 as a therapeutic host target controlling SARS-CoV-2 infection

doi: 10.1038/s44321-024-00188-x

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The lentiviral CRISPR plasmids were generated by cloning single guide RNA targeting FABP4 or control sgRNA into lentiviral CRISPR backbone lentiCRISPRv2 (Addgene, 52961).

Techniques: Knock-Out, Sequencing, Recombinant, shRNA, Staining, Electron Microscopy, Plasmid Preparation, Software, Isolation, cDNA Synthesis, Luciferase, Enzyme-linked Immunosorbent Assay