Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: Development of split luciferase complementation assay. (a) Schematic of split luciferase complementation assay. Interaction of putative interacting partners reconstitutes Gaussia luciferase, resulting in luminescence upon addition of substrate. (b) Calculation of normalized luminescence ratio (NLR). (c) luc-tagged polymerase components were transfected into HEK 293T cells along with chANP32A luc1 and the remaining polymerase components. (d and e) HEK 293T cells were transfected with expression plasmids encoding PB1 luc1 , chANP32A luc2 , PB2 627E, PA, and 0, 6.25, 12.5, or 25 ng untagged PB1 (d) or untagged chANP32A (e). (f) Minigenome components were transfected into HEK 293T cells including either untagged PB1 or PB1 luc1 and untagged chANP32A or chANP32A luc2 . Total DNA was kept constant between samples by addition of an empty vector. Results presented relative to untagged PB2 627E polymerase activity (leftmost black bar). Twenty-four hours after transfection, cells were lysed and luminescence was measured. Results shown are mean ± standard deviation from triplicate samples. Results representative of three independent experiments. Statistical significance was assessed by one-way analysis of variance, and comparisons were made between each condition in panel c and to the sample with 0 ng additional PB1 or chANP32A (leftmost black bar) in panels d and e. Multiple t tests were carried out for panel f. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Nonsignificant comparisons have not been annotated.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Luciferase, Transfection, Expressing, Plasmid Preparation, Activity Assay, Standard Deviation

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: Interaction with the polymerase is greater with chANP32A than with huANP32A. (a) PB1 luc1 , PB2 627E, PA, and either chANP32A luc2 or huANP32A luc2 were expressed in HEK 293T cells. Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Results shown are mean ± standard deviation from triplicate samples. (b) Expression plasmids encoding PB1, PB2 627E, PA, and either chANP32A-FLAG, huANP32A-FLAG, or GFP-FLAG were transfected into HEK 293T cells. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 627E was detected using Western blotting. IN, input; PD, pulldown. (c) Minigenome components were transfected into eHAP1 cells in which huANP32A and -B had been knocked out. Conditions included either untagged PB1 or PB1 luc1 and untagged huANP32A or huANP32A luc2 . huANP32A was replaced with GFP for use as a negative control. PB2 627K was used under all conditions. Total DNA was kept constant between samples by addition of an empty vector. Results are represented relative to untagged constructs (left black bar). (d and e) PB1 luc1 , PA, and either chANP32A luc2 (d) or huANP32A luc2 (e) were expressed with the addition or absence of PB2 627E. Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Western blot assay to show expression of tagged plasmids is shown beneath. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. Statistical significance was assessed by Student’s t test (*, P < 0.05; ***, P < 0.001). Results representative of three independent experiments.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Transfection, Standard Deviation, Expressing, Immunoprecipitation, Western Blot, Negative Control, Plasmid Preparation, Construct, Control, Luciferase

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: Interactions between the polymerase and ANP32 are localized in the nucleus. (a) Schematic of BiFC assay. Interaction of putative binding partners reconstitutes Venus, resulting in fluorescence. (b) HEK 293T cells were transfected with expression plasmids encoding PB1 VN , PA, and either chANP32A VC or huANP32A VC in the presence or absence of PB2 627E. Twenty-four hours after transfection, cells were fixed and fluorescence was measured by confocal microscopy. Bars, 10 μm.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Bimolecular Fluorescence Complementation Assay, Binding Assay, Fluorescence, Transfection, Expressing, Confocal Microscopy

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: Interactions require the 627 domain of PB2 and the LCAR domain of chANP32A. (a) HEK 293T cells were transfected with expression plasmids encoding PB1 luc1 , PA, either chANP32A luc2 or huANP32A luc2 , and either PB2 627E or PB2 Δ535–667. Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Statistical significance was assessed by multiple t tests. (b and c) chANP32A-FLAG (b) or huANP32A-FLAG (c) was expressed in HEK 293T cells with PB1, PA, and either PB2 627E or PB2 Δ535–667. GFP-FLAG was used as a control in place of ANP32-FLAG. Thirty hours after transfection, cell were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. IN, input; PD, pulldown. (d) Schematic of chANP32A mutants. NLS, nuclear localization signal. (e) HEK 293T cells were transfected with expression plasmids encoding PB1, PB2, PA, and either chANP32A-FLAG, chANP32AΔ4-FLAG, chANP32AΔ33-FLAG, chANP32A 1–208-FLAG, or GFP-FLAG. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. (f) HEK 293T cells were transfected with expression plasmids encoding the heterotrimeric polymerase subunits and NP and a PolI plasmid expressing an influenza virus-like RNA as well as either chANP32A-FLAG, chANP32AΔ4-FLAG, chANP32AΔ33-FLAG, chANP32A 1–208-FLAG, or an empty plasmid. Expression of Renilla luciferase was used as an internal control. Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Results shown are mean ± standard deviation from triplicate samples. Results representative of three independent experiments. Statistical significance was assessed by one-way analysis of variance, and comparisons were made to chANP32A (black bar). ns, nonsignificant; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Transfection, Expressing, Control, Immunoprecipitation, Western Blot, Plasmid Preparation, Virus, Luciferase, Standard Deviation

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: The PB2 E627K mutation does not significantly increase interaction with huANP32A. (a) Expression plasmids encoding chANP32A luc2 or huANP32A luc2 were transfected into HEK 293T cells with PB1 luc1 , PA, and either PB2 627E (black bars) or PB2 627K (gray bars). Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Statistical significance was assessed by multiple t tests and comparisons between PB2 627E and PB2 627K were made. (b) chANP32A-FLAG or huANP32A-FLAG was expressed in HEK 293T cells with PB1, PA, and either PB2 627E or PB2 627K. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. IN, input; PD, pulldown. (c) HEK 293T cells were transfected with expression plasmids encoding PB1 VN , PA, either PB2 627E or PB2 627K, and either chANP32A VC or huANP32A VC . Twenty-four hours after transfection, cells were fixed and mean fluorescence intensity (MFI) was quantified by flow cytometry. Different colors represent three independent experiments. Statistical significance was assessed using one-way analysis of variance. *, P < 0.05. (d) eHAP1 control (WT) or double-knockout (dKO) cells were transfected with expression plasmids encoding PB1 luc1 , PA, huANP32A luc2 , and either PB2 627E (black bars) or PB2 627K (gray bars). Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Results presented relative to luminescence from 627E-containing samples. (e) eHAP1 WT or dKO cells were transfected with expression plasmids encoding PB1, PB2 627E or PB2 627K, PA, and either huANP32A-FLAG or GFP-FLAG. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. Statistical significance was assessed by two-way analysis of variance, and comparisons were made between PB2 627E and 627K. ns, nonsignificant; *, P < 0.05. Results representative of three independent experiments.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Mutagenesis, Expressing, Transfection, Immunoprecipitation, Western Blot, Fluorescence, Flow Cytometry, Control, Double Knockout

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: The ANP32-polymerase interaction is stabilized at RNPs. (a to c) HEK 293T cells were transfected with expression plasmids encoding PB2 627E, PA, chANP32A luc2 , PB1 D446Y luc1 (a) or PB1 luc1 (b and c), and 0, 10, 100, 200, 300, or 400 ng of either a PolI plasmid expressing an influenza virus-like vRNA of 76 nt in length (a), a PolI plasmid expressing an influenza virus-like vRNA of 1,723 nt in length (b), or a PolI plasmid expressing an influenza virus-like cRNA of 1,723 nt in length (c). Twenty-four hours after transfection, cells were lysed, and luminescence was measured. Western blotting assay to show expression of tagged plasmids is shown beneath the graphs. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. (d and e) Cells were transfected with PB1 D446Y luc1 , huANP32A luc2 , PA, 400 ng of a PolI plasmid expressing a 1,723-nt vRNA or cRNA, and either PB2 627K (d) or PB2 627E (e). Statistical significance was assessed by one-way analysis of variance, and comparisons were made to sample with no added RNA (left black bar). ns, nonsignificant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Results representative of three independent experiments.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Transfection, Expressing, Plasmid Preparation, Virus, Western Blot, Control, Luciferase

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: The interaction between ANP32A and polymerase subsides on an active polymerase. (a to c) HEK 293T cells were transfected with PB1 luc1 ; PA; 0, 0.625, 1.25, 2.5, 5, or 10 ng PolI 76-nt vRNA; and either chANP32A luc2 and PB2 627E (a), huANP32A luc2 and PB2 627K (b), or huANP32A luc2 and PB2 627E (c). Western blotting assay to show expression of tagged plasmids is shown beneath the graphs. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. Statistical significance was assessed by one-way analysis of variance, and comparisons were made to sample with no added RNA (leftmost bar). ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (d) HEK 293T cells were transfected with expression plasmids encoding PB1, PB2 627E or PB2 627K, PA, and either chANP32A-FLAG or huANP32A-FLAG in the presence or absence of a PolI plasmid expressing a viral-like RNA. GFP-FLAG was used as a control. Thirty hours after transfection, cells were lysed, FLAG-tagged proteins were immunoprecipitated, and coprecipitation of PB2 was detected using Western blotting. IN, input; PD, pulldown. Results representative of three independent experiments.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Transfection, Western Blot, Expressing, Control, Luciferase, Plasmid Preparation, Immunoprecipitation

Journal: Journal of Virology

Article Title: Elucidating the Interactions between Influenza Virus Polymerase and Host Factor ANP32A

doi: 10.1128/JVI.01353-19

Figure Lengend Snippet: huANP32A is still required for replication of a vRNA template with 3′5′8 mutations. (a and b) WT or dKO eHAP1 cells were transfected with minigenome assay components containing a polymerase bearing either PB2 627E (a) or PB2 627K (b) and PolI plasmids expressing either wild-type vRNA (black bars) or with the 3′5′8 mutations (gray bars). Results were normalized to Renilla luciferase and presented relative to control cells (WT). (c) eHAP1 dKO cells were transfected with minigenome assay components using only polymerase containing PB2 627E. Either 40 ng empty plasmid or 20 ng huANP32A and 20 ng huANP32B were also expressed. Results were normalized to Renilla luciferase and presented relative to dKO empty plasmid. Statistical significance was assessed by multiple t tests. ns, nonsignificant; **, P < 0.01; ****, P < 0.0001. (d and e) 293T cells were transfected with either PB2 627K (d) or PB2 627E (e) and PB1 luc1 , PA, and 0, 0.625, 1.25, 2.5, 5, or 10 ng PolI 76-nt vRNA. (f) Cells were transfected with PB1 D446Y luc1 , ANP32A luc2 , PA, and either no RNA, 400 ng PolI 76-nt vRNA, or 76-nt vRNA mut3′5′8. Western blotting assay to show expression of tagged plasmids is shown beneath the graphs. Vinculin was used as a loading control. Gaussia luciferase antibody recognizes both luc1 and luc2. Statistical significance was assessed by one-way analysis of variance, and comparisons were made to sample with no added RNA (black bars). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Results representative of three independent experiments.

Article Snippet: HEK 293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS) (Biosera), 1% penicillin-streptomycin (Pen-Strep) (Gibco), and 1% nonessential amino acids (Sigma-Aldrich).

Techniques: Transfection, Expressing, Luciferase, Control, Plasmid Preparation, Western Blot

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Effect of peritoneal dialysis solution (PDS) with and without N-acetylcysteine (NAC) on drain volume (A), plasma glucose (B), and adiponectin levels in plasma (C) and spent dialysate (D) in rat. After 12 weeks of PD, a peritoneal equilibrium test using 3.86% glucose PDS was performed immediately before the rats were sacrificed; plasma and spent dialysate were collected and analyzed. Values are mean ± SE of 8 animals. *p < 0.05 versus sham, †p < 0.05 versus PDS-treated rats.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Clinical Proteomics

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Differentiation of 3T3-L1 preadipocytes. Images of mouse cells were captured on days 0, 4, and 10 by light microscope. Inset pictures represent Oil Red O staining. 3T3-L1 cells were fully differentiated at day 10 (A; right panel). Adiponectin mRNA expression level during 3T3-L1 cell differentiation was examined by RT-PCR (B). Adiponectin secretion in the media was measured by ELISA after 24-hour incubation under indicated experimental condition (C). Relative secretion was calculated as the ratio of adiponectin to protein (μg/mg). Basal adiponectin secreted in the media was 6.0 ± 0.7 μg/mg protein. Values are mean ± SE of 4 – 5 independent experiments. *p < 0.05 versus control; †p < 0.05 versus H2O2 200 μmol/L. HG = high D-glucose; PDS = peritoneal dialysis solution diluted twofold with DMEM; RT-PCR = reverse-transcription polymerase chain reaction.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Light Microscopy, Staining, Expressing, Cell Differentiation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Peritoneal Dialysis International : Journal of the International Society for Peritoneal Dialysis

Article Title: Glucose-Based Peritoneal Dialysis Solution Suppresses Adiponectin Synthesis Through Oxidative Stress in an Experimental Model of Peritoneal Dialysis

doi: 10.3747/pdi.2009.00228

Figure Lengend Snippet: — Effect of H2O2 on adiponectin oligomer secretion (A), adiponectin mRNA expression during 3T3-L1 cell differentiation (B), and in adipocytes freshly isolated from mouse abdominal fat pads (C). The effect of H2O2 (H; 200 μmol/L) on multimer, hexamer, and trimer adiponectin secretion was evaluated under nonreducing conditions. mRNA expression levels were examined by RT-PCR and real-time PCR. Values are mean ± SE of 3 – 4 independent experiments. *p < 0.05 versus control (C) at each time point. RT-PCR = reverse-transcription polymerase chain reaction; HMW = high-molecular weight 12- to 18-mer; MMW = middle-molecular weight hexamer; LMW= low-molecular weight trimer.

Article Snippet: A commercial ELISA kit for rat adiponectin (R&D Systems, Minneapolis, MN, USA) was used to quantify adiponectin in plasma and spent dialysate and a kit for mouse adiponectin (R&D Systems) was used for 3T3-L1 adipocyte culture media according to manufacturers’ instructions.

Techniques: Expressing, Cell Differentiation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction, High Molecular Weight, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Bacterial Strains and Plasmid Constructs used in this study.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Plasmid Preparation, Construct, Control, Knock-In, Over Expression, Recombinant

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: B. miyamotoi encodes two orthologous genes to B. burgdorferi BBK32. (A) BBK32 orthologs are found in relapsing fever (RF)-associated and B. miyamotoi spirochetes and are denoted FbpA, FbpB, and FbpC . B. miyamotoi FR64b FbpA and FbpB are underlined. (B) An alignment of B. miyamotoi strain FR64b FbpA and FbpB to B. burgdorferi strain B31 BBK32 shows differences of the amino acid sequences within the fibronectin binding (green box) and complement inhibitory domains (blue box). The gelatin-binding domain (GBD) of BBK32 is denoted. The key residues R248 and K327 of BBK32 involved in complement C1r binding are indicated by a red box. * conserved, : strongly similar, . weakly similar.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Assessing the interaction of human C1r with B. miyamotoi FbpA and FbpB. SPR was used to assess protein-protein interactions between the C-terminal regions of each Fbp protein and activated C1r-CCP2-SP. Immobilized (A) FbpA-C, (B) FbpB-C, and (C) FbpA-C-R264A-K343A (referred to as FbpA DA-C throughout) were subjected to an injection series of serially diluted C1r-CCP2-SP (0.78 - 100 nM). A representative sensorgram from the three replicates for each Fbp is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D values were determined using kinetic fits and are shown as the mean +/- standard deviation of three replicates in Table 4 . A K D value for C1r-CCP2-SP interactions with FbpA DA-C was not determined (N.D.). (D, E) Competition experiments were carried out using 25 nM C1r-CCP2-SP alone or mixed with 25 nM soluble Fbp proteins injected over FbpA-C (D) or FbpB-C (E) . (F) Comparisons of statistical significance were performed for data shown in panels (D, E) using a one-way ANOVA followed by a multiple comparison Tukey test (* = p < 0.05).

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Protein-Protein interactions, Injection, Standard Deviation, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: SPR and complement assay results.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Complement Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: FbpA-C and FbpB-C interact differentially with zymogen and activated forms of human C1r. (A) Single cycle SPR was used to determine binding affinities of C1r zymogen or active C1r ranging from 0.16 - 100 nM injected over immobilized Fbps. A representative sensorgram from a three-injection series for each Fbp-C and C1r active state is shown with the black curve being the sensorgram and the red curve the associated kinetic fit. K D ’s were determined using kinetic fits and are shown as the average and standard deviation of three replicates in Table 4 . (B) Far Western overlays using B. burgdorferi B314 lysates probed with zymogen or enzymatic forms of purified full-length C1r, followed by an antibody to C1r. (C) An ELISA-type binding assay was used to determine the ability of FbpA-C, FbpA DA-C and FbpB-C to interact with C1r in human serum. FbpA-C (black), FbpB-C (blue), and FbpA DA-C (red) were immobilized on an ELISA plate then incubated with a two-fold dilution series of normal human serum ranging from 0.0012-10%. Corresponding EC 50 values for FbpA-C, FbpB-C, and FbpA DA-C are reported in Table 4 . (D) Fbp-mediated inhibition of purified active C1r-CCP2-SP enzymatic activity was assessed by incubating 15 nM C1r-CCP2-SP with a 1 μM of FbpA-C (white), FbpB-C (striped), and FbpA DA-C (grey). C1r-CCP2-SP enzymatic activity was determined by incubation with a C1r substrate (Z-Gly-Arg-sBzl) that, once cleaved, reacts with DTNB resulting in a colorimetric change. No statistical difference was found between FbpA-C and FbpB-C inhibitory activity, whereas FbpA DA-C exhibited significant loss in inhibitory activity. Statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Tukey test (*=p<0.05).

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Binding Assay, Injection, Standard Deviation, Western Blot, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Inhibition, Activity Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Borrelia miyamotoi FbpA and FbpB Are Immunomodulatory Outer Surface Lipoproteins With Distinct Structures and Functions

doi: 10.3389/fimmu.2022.886733

Figure Lengend Snippet: Determining the ability of surface-expressed B. miyamotoi FbpA and FbpB to protect a serum-sensitive strain of B. burgdorferi . (A) Western blots of lysates from B. miyamotoi strain FR64b and B burgdorferi B314 isolates expressing fbpA , fbpA-DA , fbpB , and bbk32 were probed with antibodies to FbpA (anti-FbpA), FbpB (anti-FbpB), BBK32 (anti-BBK32) and FlaB (anti-FlaB), the latter as a loading control and a control for a subsurface target. Vector refers to the plasmid-only backbone sample (B314/pBBE22 luc ). Samples from each population were subjected to proteinase K accessibility treatments to determine the surface expression of the proteins. Due to its periplasmic location, the flagellar protein, FlaB, is unaffected by proteinase K in intact cells and depicts structural integrity in the treated B. miyamotoi and B314 derivatives listed. (B) B. miyamotoi Fbp proteins were tested for their ability to confer resistance to normal human serum (NHS) sensitive B. burgdorferi strain B314 relative to vector-only and BBK32-expressing controls (negative and positive controls, respectively). Asterisks depict a significant increase in survival relative to FbpA DA and the vector control (*=p<0.0001). All strains exposed to heat inactivated NHS (hiNHS), rendering complement proteins inactive, were largely unaffected. (C) Rescue assays were used to determine if soluble exogenous recombinant proteins could promote survival of serum-sensitive vector-containing B. burgdorferi B314. Increasing five-fold concentrations of BBK32-C, FbpA-C, FbpA DA-C, and FbpB-C were added in serum sensitivity assays. The middle samples (i.e., 240 nM) have added recombinant protein that is roughly equivalent to the concentration of C1r in the assay conditions employed. Cells were assessed via darkfield microscopy. Included as controls are a vector-only strain of B314 with no exogenous protein addition, as well as a BBK32-producing B314 isolate (right side). Asterisks depict a significant increase in survival relative to the vector control (*=p<0.015). Significance for serum sensitivity and rescue assays was determined using two-way ANOVA with a Šidák correction for multiple comparisons.

Article Snippet: The amount of serum C1r bound to immobilized Fbps was found using a goat antibody to human C1r (R&D Systems) diluted 1:2,000 and immune complexes detected with rabbit anti-goat Ig conjugated to HRP (Invitrogen) at a 1:3,000 dilution.

Techniques: Western Blot, Expressing, Control, Plasmid Preparation, Recombinant, Concentration Assay, Microscopy

Journal: Brain Communications

Article Title: Clusterin accumulates in synapses in Alzheimer’s disease and is increased in apolipoprotein E4 carriers

doi: 10.1093/braincomms/fcz003

Figure Lengend Snippet: Clusterin is increased in the synaptic compartment in Alzheimer's disease with highest levels in APOE4 carriers. Western blot ( A , B ) analysis shows an increased level of clusterin when comparing Control with Alzheimer's disease in both Crude Homogeante ( A , C ) and in Synaptically enriched preps ( B , D ). In synapses, there is a further increase in the Alzheimer's disease APOE4 cases compared with Alzheimer's disease APOE3 cases (*Tukey’s post hoc tests, P < 0.05).

Article Snippet: Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hemel Hempstead, UK), which were probed with the following primary antibodies: β-actin (ab8226, Abcam, 1:2000), Synaptophysin (ab8049, Abcam, 1:10 000), GAPDH (ab8245, Abcam, 1:2000), Histone H3 (1:1000, ab1791, Abcam), PSD95 (1:1000, D27E11, Cell Signaling Technology) and clusterin (1:500, sc-8354, Santa Cruz Biotechnology).

Techniques: Western Blot, Control

Journal: Brain Communications

Article Title: Clusterin accumulates in synapses in Alzheimer’s disease and is increased in apolipoprotein E4 carriers

doi: 10.1093/braincomms/fcz003

Figure Lengend Snippet: Representative images of array tomography staining. Array tomography ribbons from non-demented controls (NDC), Alzheimer's disease APOE3 and Alzheimer's disease APOE4 individuals were stained for pre-synapses (synaptophysin, yellow), oAβ (1C22, cyan) and clusterin (magenta). Images shown in ( A ) are maximum intensity projections of four serial sections (aligned raw images). Images shown in ( B ) are maximum intensity projections of two serial sections from analysed image stacks that have been thresholded and single section noise removed in MATLAB. Each channel is shown separately with the merge in the bottom image. Arrows indicate synapses containing both clusterin and oAβ staining. Scale bars represent 15 μm ( A ) and 1 μm ( B ).

Article Snippet: Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hemel Hempstead, UK), which were probed with the following primary antibodies: β-actin (ab8226, Abcam, 1:2000), Synaptophysin (ab8049, Abcam, 1:10 000), GAPDH (ab8245, Abcam, 1:2000), Histone H3 (1:1000, ab1791, Abcam), PSD95 (1:1000, D27E11, Cell Signaling Technology) and clusterin (1:500, sc-8354, Santa Cruz Biotechnology).

Techniques: Tomography, Staining

Journal: Brain Communications

Article Title: Clusterin accumulates in synapses in Alzheimer’s disease and is increased in apolipoprotein E4 carriers

doi: 10.1093/braincomms/fcz003

Figure Lengend Snippet: Analysis of synaptic punctate by array tomography. ( A )There is a significant decrease in the pre-synaptic density in Alzheimer’s cases near plaques (<10 μm) compared with far from plaques (>45 μm) which is exacerbated by APOE4 genotype (one-way ANOVA F [4, 22] = 14.2, P < 0.0001). ( B ) There is also a significant increase in the percent of Aβ (1C22) positive synapses near plaques compared with far from plaques that is most pronounced in E4 cases (Kruskal–Wallis test, P = 0.0003). ( C ) Clusterin in pre-synaptic terminals is increased in Alzheimer's disease APOE4 carriers (Kruskal–Wallis, P = 0.0003). ( D ) Similarly, the percentage of synapses containing both clusterin and Aβ is highest in Alzheimer's disease APOE4 carriers near plaques carriers (Kruskal–Wallis, P = 0.0002). (* P < 0.05, ** P < 0.01, **** P < 0.0001 Tukey’s post hoc test. # P < 0.05, ## P < 0.01, ### P < 0.001 post hoc Mann–Whitney U .) Each symbol represents the mean for a single case in ( A ) with error bars representing standard deviation. Each symbol in ( B–F ) represents the median for a single case with the error bars showing inter-quartile ranges.

Article Snippet: Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hemel Hempstead, UK), which were probed with the following primary antibodies: β-actin (ab8226, Abcam, 1:2000), Synaptophysin (ab8049, Abcam, 1:10 000), GAPDH (ab8245, Abcam, 1:2000), Histone H3 (1:1000, ab1791, Abcam), PSD95 (1:1000, D27E11, Cell Signaling Technology) and clusterin (1:500, sc-8354, Santa Cruz Biotechnology).

Techniques: Tomography, MANN-WHITNEY, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-β, Collagen Type I, and PD-L1/PD-1.

doi: 10.4049/jimmunol.1800717

Figure Lengend Snippet: FIGURE 7. Effect of LOAd viruses in vivo. (A) C57BL6/J mice (n = 10 per group) were injected with syngeneic B16 melanoma expressing human CD46 to enable virus infection. At day 5, the tumor was visible under the skin, and the mice were treated by s.c. injection in the tumor area with a LOAd virus expressing murine TMZ-CD40L (mLOAd700; 1 3 109 infection particles per mouse in 50 ml) and/or a rat anti-mouse IL-6Ra Ab (0.5 mg/mouse in 50 ml) or treated with NaCl control. The mice were treated twice a week, six injections in total. Survival was determined by log-rank test, and mLOAd700 was significantly different from control (p = 0.0145). (B) At day 13 (1 d after third treatment), five mice per group were sacrificed, and the tumors were analyzed for immune cell populations using flow cytometry. Statistical differences were calculated by Kruskal–Wallis (ANOVA) with Dunn multiple comparison test. Statistical significance is indicated with *p , 0.05.

Article Snippet: A virus expressing murine TMZ-CD40L (mLOAd700) was injected s.c. in the tumor area (1 3 109 infectious particles per mouse in 50 ml) with or without coinjection of a rat anti-mouse IL-6Ra Ab (0.5 mg/mouse in 50 ml; BioXCell, West Lebanon, NH).

Techniques: In Vivo, Injection, Expressing, Virus, Infection, Control, Cytometry, Comparison

Journal: PLoS ONE

Article Title: Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

doi: 10.1371/journal.pone.0092608

Figure Lengend Snippet: ( A ) Schematic representation of the differentiation program indicating the time course of treatments. ( B ) Lipid accumulation was assessed by Oil-Red-O staining (magnification: 10× for second panel). ( C ) Quantification of Oil-Red-O positive lipid droplets for each condition from eight random microfields from three independent biological replicates. Error bars represent the standard error of the mean. ( *p<0.05 ) ( D ) Immunoblot analyses for key pan-adipogenic markers, ( E ) Q-PCR analyses of pan-adipogenic markers ( Pparγ, Fabp4, Adiponectin, Hormone sensitive lipase, Perilipin 4, Lipoprotein lipase ), ( F ) white adipose tissue markers ( Hoxc9, Psat1 ), myogenic markers ( Myod1, Myogenin ) and osteogenic marker ( Runx2 ) in cells pretreated with or without BMP6 or BMP7 (250 ng/mL) followed by adipogenic differentiation. ( G ) Q-PCR analyses of pan-adipogenic markers in cells treated with or without 250 ng/mL BMP8 followed by adipogenic differentiation. Expression in untreated cells was set to 1 and results represent triplicate analyses of three independent biological replicates (mean ± SD), *p<0.05 versus no BMP. Similar results were obtained in at least 2–3 independent analyses.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: Optn (sc-166576), Gapdh (sc-32233) and the following were purchased from Cell Signaling: Pparγ (2435S), Fabp4 (3544S), Adiponectin (2789S), and Cox2 (12282S).

Techniques: Staining, Western Blot, Marker, Expressing

Journal: PLoS ONE

Article Title: Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

doi: 10.1371/journal.pone.0092608

Figure Lengend Snippet: ( A ) Q-PCR analyses of BAT thermogenic markers ( Ucp-1 , Dio2 and Pgc-1α ) and BAT specific markers ( Cidea and Elovl3 ) in BMP6 or BMP7 (250 ng/mL) pretreated cells followed by adipogenic differentiation and stimulation with or without 10 μM forskolin for 4 hours. ( B ) Parallel analyses of BAT thermogenic markers in BMP8 (250 ng/mL) pretreated cells. The expression in untreated cells (no BMP pretreatment and no forskolin stimulation: no BMP-no F) was set to 1, and results represent triplicate analyses of three independent biological replicates (mean ± SD), *p<0.05 versus no BMP-F or no BMP, Student's t test. Similar results were obtained in at least two independent analyses. ( C ) Q-PCR generated Ct values for Prdm16 and Gapdh transcripts at the end of differentiation cascade following BMP6 or BMP7 stimulation. ( D ) Ct values for Prdm16 and Gapdh in relevant phases of differentiation landscape following BMP6 stimulation. Each dot per phase represents an independent biological replicate. For all the Q-PCR analyses, Gapdh served as the endogenous control. ( E ) Heat map depiction and unbiased clustering analyses of 1718 significantly changing qualifiers ( p≤0.05, fold change≥1.5 ) in C2C12 cells stimulated with or without BMP6 for two days followed by differentiation protocol outlined in . Key brown fat and pan-adipogenic markers: Fabp4, Adiponectin, Fabp5, Monoglyceride lipase, Dio2, Lipoprotein lipase (Lpl), Perilipin 4, Pparγ, and CD36, and myogenic markers : Calsequestrin 2 (Casq2), Myozenin 1 (Myoz1), Tropomyosin 3 (Tpm3), Myosin binding protein C-fast type (Mybpc2) and Myosin regulatory light chain 2 (Myl2) are highlighted in the heat map. Each gene is represented by a single row and each sample in three independent biological replicates for each time point, by a column. Two distinct clusters indicate genes induced (red) and repressed (green), with representative induced and repressed genes highlighted in red and green boxes respectively.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: Optn (sc-166576), Gapdh (sc-32233) and the following were purchased from Cell Signaling: Pparγ (2435S), Fabp4 (3544S), Adiponectin (2789S), and Cox2 (12282S).

Techniques: Expressing, Generated, Control, Binding Assay

Journal: PLoS ONE

Article Title: Brown Fat Determination and Development from Muscle Precursor Cells by Novel Action of Bone Morphogenetic Protein 6

doi: 10.1371/journal.pone.0092608

Figure Lengend Snippet: ( A ) Transcript knockdown validation of commitment phase candidate genes using Q-PCR (mean ± SD), * p<0.05. ( B ) Q-PCR validation for Ucp-1 transcript levels in cells transduced with or without short hairpin constructs against indicated genes post two days BMP6 pretreatment followed by adipogenic differentiation and 10 uM forskolin stimulation. The expression in parental cells in the absence of forskolin stimulation was set to 1. A reduction in Ucp-1 transcript levels by more than 40% of levels observed in forskolin stimulated parental cells was set as the lower threshold to choose the gene(s) of interest for subsequent investigations. Results represent triplicate analyses of two to three independent biological replicates (mean ± SD). ( C ) Bar graph representation of Optn and Cox2 transcript levels at indicated time points measured using Affymetrix array. ( D ) Verification of Optn and Cox2 induction at the protein level by immunoblot analyses at indicated time points post BMP6 stimulation. Gapdh served as the loading control. ( E ) Knockdown of Optn and Cox2 was validated using immunoblot analyses in basal state and after two days of BMP6 stimulation. ( F ) Q-PCR validation for BAT markers ( Cidea, Cox7a1 ) showing reduced induction in the context of attenuated Optn and Cox2 expression in indicated cells. Expression in Optn and Cox2 proficient cells was set to 1 and Gapdh served as the endogenous control. Results represent triplicate analyses of three independent biological replicates and similar results were obtained in at least two independent analyses (mean ± SD), * p<0.05 . ( G ) Immunoblot analyses for pan-adipogenic markers (PPARγ, Fabp4 and Adiponectin). Quantification of band intensity is provided above the respective blot. Note that the quantification data provided for PPARγ blot corresponds to the intensity of PPARγ2 (top band, panel G). ( H ) Two representative images for Oil-Red-O staining in Optn and Cox2 proficient and knockdown cells are shown. Analyses for panels F, G and H was performed at the end of the differentiation cascade illustrated in following two days of BMP6 (250 ng/mL) stimulation. Also see and .

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology: Optn (sc-166576), Gapdh (sc-32233) and the following were purchased from Cell Signaling: Pparγ (2435S), Fabp4 (3544S), Adiponectin (2789S), and Cox2 (12282S).

Techniques: Knockdown, Biomarker Discovery, Transduction, Construct, Expressing, Western Blot, Control, Staining

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 1. Adiponectin is the most enriched adipokine in VAT-derived EVs (A and B) Lean (Ctrl) and obese (Ob) VAT-derived lEV and sEV mode size comparison. Size-distribution curves (A) and mean mode size (B). Ctrl and Ob EV subtype size-distribution curves (A) are represented by plain or dashed lines, respectively, and are presented as the mean ± SEM (n = 4–6 independent EV preparations for each condition). (C and D) EV marker analysis from lean (Ctrl) or ob/ob (Ob) VAT explant-derived EV subpopulations. A significant enrichment in CD63 was observed for obese VAT-derived sEVs (D). (E) lEV and sEV secretion from VAT, SAT, and BAT explants. EV number secreted per gram of AT per 24 h is presented. (F) Increased lEV and sEV secretion from mouse VAT with obesity. EV secreted by total mouse VAT is presented. (G) EV secretion of human omental, mesenteric (Mesent.), and subcutaneous (Subcut.) AT collected from obese subjects. Secretion of EVs is presented as the number of EVs secreted per gram of human AT.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Comparison, Marker

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 3. Plasma EVs represent stable carriers of adiponectin (A) VAT-derived EVs are retrieved in the blood circulation, as illustrated by flow-cytometry detection of ZsGreen+ EVs in platelet-free plasma (PFP). Ctrl, control; AdipoZS1, Cre AdipoZs1 mice; AdipoZS1, Cre+ AdipoZs1 mice. (B) Adiponectinemia significantly decreases upon plasma EV removal in lean mice. (C) The presence of adiponectin was confirmed in mouse plasma circulating EVs. Plasma EV subtypes were isolated from lean and obese mice. Ten micrograms of each EV subtype was resolved by SDS-PAGE under reducing conditions (R) or nonreducing unheated conditions (NR). One representative blot (out of two experiments performed) is presented. (D) Adiponectin clearance measurement following the injection of serum or EV-depleted serum in adiponectin KO mice. n = 6 mice injected per group. (E) Western blot of human plasma sEVs confirms the decreased amount of adiponectin in sEVs isolated from obese patients compared to control patients. A representative blot (out of three independent experiments performed) is presented with quantification of the Adpn blot signal intensity.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Clinical Proteomics, Derivative Assay, Cytometry, Control, Isolation, SDS Page, Injection, Western Blot

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 4. EV-associated adiponectin maintains insulin sensitivity in target cells (A and B) Adiponectin-enriched sEVs reverse insulin resistance in hepatocytes. Ctrl sEVs and Ob sEVs correspond to VAT-derived sEVs isolated from either lean or obese VAT, respectively. Adpn-Ob sEVs refer to the enrichment of Ob sEVs with adiponectin (following their preincubation with EV-free conditioned media). Ins, insulin; Palm, palmitate. (C) Silencing of both AdipoR1 and AdipoR2 significantly reduces the VAT-derived sEV insulin-sensitizing effects. Scr, Scramble; R1, AdipoR1; R2, AdipoR2. (D) Rapid and time-dependent internalization of fluorescent adiponectin-Venus (Adpn-Venus) internalization in hepatocytes. Scale bars, 50 mm. (E) Adiponectin enrichment in sEVs is responsible for their insulin-sensitizing effects, independent of their sEV cellular origin. Dot plots represent independent experiments. Data are presented as the mean ± SEM. Statistical differences were assessed using one-way ANOVA. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Derivative Assay, Isolation

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 5. EV-associated adiponectin reverses HFD-induced insulin resistance in mice (A) NHS ester-labeled VAT-derived sEV organ biodistribution. Licor fluorescent tissue imaging is presented in Figure S4A. (B) Adoptive transfer of sEV-associated adiponectin limits weight gain induced by a high-fat diet (HFD). A standard diet (SD) is presented as a control to demonstrate HFD-induced weight gain compared to body weight on a regular chow diet. Number of animals per group: SD, n = 3; NaCl, n = 15; Ctrl sEVs, n = 13; AdpnKO sEVs, n = 11. (C) Tissue weights at sacrifice of mice that received i.p. injections of Ctrl sEVs, AdpnKO sEVs, or NaCl (vehicle) over the course of 5 weeks of HFD compared to mice maintained on an SD.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Labeling, Derivative Assay, Imaging, Adoptive Transfer Assay, Control

Journal: Cell reports

Article Title: Extracellular vesicles are carriers of adiponectin with insulin-sensitizing and anti-inflammatory properties.

doi: 10.1016/j.celrep.2023.112866

Figure Lengend Snippet: Figure 6. EV-associated adiponectin dis- plays anti-inflammatory properties (A) Macrophage (Mac2) staining in SAT (upper panels) and VAT (lower panels) depots from sEV-injected mice reveal the anti-inflammatory properties of Ctrl sEVs treatment. HFD-induced adipocyte hypertrophy and HFD-related macro- phage infiltration are observed in both SAT and VAT compared to SD-fed conditions. Scale bars, 50 mm. (B and C) qPCR mRNA expression of inflammatory markers in VAT (B) and liver (C) tissues. Dot plots represent the number of independent animals analyzed. Data are presented as the mean ± SEM. Statistical differences were calculated compared to the HFD + NaCl group using one-way ANOVA followed by the Kruskal-Wallis test. Statistical significancy is indicated for each panel as follows : *p % 0.05, **p % 0.01, ***p % 0.01,****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Adiponectin (murine) Thermofisher Scientific #PA1-054, RRID:AB_325789 Adiponectin (human) Abeomics #10-7597, RRID:AB_2943077 AKT pan (40D4) Cell signaling Cat #2920, RRID:AB_1147620 AKT pan polyclonal (in vivo studies) Cell signaling Cat #4691, RRID:AB_915783 Phospho-AKT (pAKT, Ser 473) Cell signaling Cat #4060S, RRID:AB_2315049 Alix BD Biosciences Cat #611620, RRID:AB_399062 Murine CD9 BD Pharmingen Cat #553758, RRID:AB_395032 human CD9 Santa Cruz Cat #SC-13118, RRID:AB_627213 Murine CD63 MBL Cat #D263-3, RRID:AB_1278815 Flotillin-2 BD Pharmingen Cat #610383, RRID:AB_397766 Mac2 Cedarlane Cat #CL8942AP, RRID:AB_10060357 MIF (human) R&D systems Cat #mab289, RRID:AB_2281975 Syntenin-1 Abcam Cat #ab19903, RRID:AB_445200 Zs-Green Clontech Cat #632598, RRID:AB_2943078 Goat anti-rat IgG2a Secondary antibody HRP conjugate (used for MaC2 staining) Invitrogen Cat# PA1-84709, RRID:AB_933942 Anti-adiponectin FITC coupled antibody Assay-Pro Cat #10361-05041, RRID:AB_2943079 IRDye 800CW and 680RD secondary antibody (anti-mouse and anti-rabbit) LI-COR Biosciences Cat #926–32211, RRID:AB_621843; Cat #926–32210,RRID:AB_621842; Cat #926–68071, RRID:AB_10956166; Cat #926-68070, RRID:AB_10956588 Chemicals, peptides, and recombinant proteins Bovine serum albumin (BSA) FFA free Sigma Cat #A7030 Collagenase A Roche Cat #10103586001 DAB OB Super Sensitive detection kit ImPath Cat #46538 DAB Quanto Thermofischer Scientific Cat #TA-125-QHDX Wash buffer for IHC ImPath Cat #45002 Antigen retrieval solution pH6.0 ImPath Cat #44998 Ultravision Hydrogen Peroxyde block Thermofisher Scientific Cat #TS-12-H2O2Q Goat serum Immunoreagents Cat #SP-004-VX10 IHC mounting medium CellPath Cat #SEA-1604-00A Qiazol Lysis reagent Qiagen Cat #79306 RNeasy mini kit Qiagen Cat #74104 SuperScriptII reverse Transcriptase Invitrogen Cat #18064-014 dNTP set 100mM Invitrogen Cat #10297-018 Random hexamers Invitrogen Cat #48190-011 QIAquick PCR purification kit Qiagen Cat #28106 RNaseOut Ribonuclease inhibitor Invitrogen Cat #10777-019 Maxima SYBR Green qPCR master mix 2X Thermofisher Scientific Cat #K0253 96-Well PCR plates Thermofisher Scientific Cat #AB0700 DMEM 4.5 g/L glucose Gibco Cat #41966-029 DMEM 1 g/L glucose Gibco Cat #31885-023 Elisa mouse Adiponectin/Acrp30 R&DSystems Cat #DY1119 and #DY008 (Continued on next page) 16 Cell Reports 42, 112866, August 29, 2023

Techniques: Staining, Injection, Expressing

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: Viral titers at 72hpi in (A) HT29, (B) SW1736, (C) HeLa, (D) HCT116, (E) A549. (F) TCID 50 value of ten additional genotype VII NDV strains in HeLa cells. Representative data, shown as the mean ± SD (n = 3), were analyzed with one-way ANOVA. ****, P <0.0001, (G) IFA experiments were performed with anti-HN and Hoechst at 24h and 48h after infection of NDVs with 0.1MOI, 1MOI, and 10MOI. (H) Replication of I 4 and Herts/33 in HeLa cells. Cells were infected with I 4 and Herts/33 at 0.1MOI, 1MOI, and 10MOI for 1 h at 37°C. Viral titers in the supernatant were expressed using TCID 50 value. (I) Apoptosis was detected by flow cytometry at 24h after infection at 10MOI, yielding apoptotic cells as a percentage of the total cell count. (J) The oncolytic effect of NDVs on HeLa cell line in vitro. HeLa cells were infected with 0.1MOI, 1MOI, and 10MOI at 0, 24, 36, and 48h. The cell viability was measured by CCK-8 assay and expressed as a percentage relative to the control group, and results are shown as the mean of three independent experiments. Representative data, shown as the means ± SDs (n = 3), were analyzed with two-way ANOVA. (****, P < 0.0001).

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Infection, Flow Cytometry, Cell Counting, In Vitro, CCK-8 Assay, Control

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) Schematic diagram of the cloning strategy for replacement of NP, P, and L genes between rHerts/33 and rI 4 . The construction strategy is described in the . The virulence of the different recombinant viruses was determined by measuring the ICPI in 1-day-old chickens. (B and C) TCID 50 value of the virulent strains after simultaneous replacement of NP, P, and L at 72hpi on tumor cell lines. (D) Expression of viral proteins on HeLa cells by recombinant viruses after simultaneous replacement of NP, P, and L. Western blot analysis was performed by anti-NP and anti-HN at 24h after infection with NDVs at 1MOI and 10MOI, respectively. (E and F) TCID 50 value of the virulent strains after individual gene replacement of NP, P, or L at 72hpi on tumor cell lines. (G-H) Viral proteins expression on HeLa cells by recombinant viruses after individual gene replacement of NP, P, or L. Western blot analysis was performed by anti-NP and anti-HN at 24h after infection with NDVs at 1MOI and 10MOI, respectively. Representative data, shown as the means ± SDs (n = 3), were analyzed with two-way ANOVA. ****, P <0.0001.

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Cloning, Recombinant, Expressing, Western Blot, Infection

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) Structural diagram of the NP protein. (B) Schematic diagram of the cloning strategy for exchanging the whole N-tail and the second IDR of the N-tail domain between rHerts/33 and rI 4 . The virulence of the different recombinant viruses was determined by measuring the ICPI in day-old chickens. (C, D, E, and F) TCID 50 value of recombinant strains after the whole N-tail and the second IDR of the N-tail domain is replaced at 72hpi on tumor cell lines. (G) Expression of viral proteins on HeLa cells by recombinant viruses. Western blot analysis was performed by anti-NP and anti-HN at 24h after infection with NDVs at 1MOI and 10MOI, respectively. (H) Schematic representation of the comparison of NP genes from different virulent strains by MEGA. (I) TCID 50 values of NDVs at 72hpi in HeLa cells and CEF cells after mutation of each conserved site in rHerts/33 and rI 4 . (J, K, L, M) TCID 50 values of mutant NDVs at 72hpi on tumor cell lines. (N) Expression of viral proteins on HeLa cells by virulent mutant strains. Western blot analysis was performed by anti-NP, anti-HN, and anti-β-actin at 12h after infection with NDVs at 1MOI and 10MOI, respectively. All experiments were repeated thrice, and results are expressed as mean ± SDs. Two-way ANOVA was used to evaluate the significance of differences. ****, P <0.0001.

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Cloning, Recombinant, Expressing, Western Blot, Infection, Comparison, Mutagenesis

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) HeLa cells and DF1 cells were infected with NDVs at 10MOI at 37°C for 1h and then incubated with anti-HN mouse monoclonal antibody and goat anti-mouse IgG/FITC at 4°C. After that, cells were washed and assessed by flow cytometry. (B, C, D) HeLa cells were infected with NDV (10MOI) at 37°C for 0.5h and then were collected at 0h, 1h, 2h, 4h, and 8h. Total RNA was extracted and reverse-transcribed using specific primers for gRNA (B), mRNA (C), and cRNA (D) of NDVs. Copy numbers were determined using quantitative RT-PCR. (E and F) Total cellular RNA was extracted at 12h and 24h after transfection of 1.5 μg minigenome into HeLa cells. Reverse transcription was performed using specific primers to detect genomic RNA (E) and mRNA (F) of GFP by quantitative RT-PCR. (G) Expression of GFP was detected at 24h in HeLa cells after transfecting 0.5μg or 1.5μg minigenome with anti-GFP, anti-NP, and anti-β-actin. (H) After transfection with different minigenome systems for 24h, cells were treated with 100μg/ml CHX, and then cells were harvested at 4, 8, and 12 hours. Expression of GFP and NP was detected with anti-GFP, anti-NP, and anti-β-actin. (I, J, K) HeLa cells were treated with 100μg/ml CHX for 30mins and then infected with NDV (10MOI) at 37°C for 0.5h. After that, cells were collected at 0h, 1h, 2h, 4h, and 8h. Total RNA was extracted and reverse-transcribed using specific primers for gRNA (I), mRNA (J), and cRNA (K) of NDVs. Copy numbers were determined using quantitative RT-PCR. Data are presented as means from three independent experiments. Significance is analyzed by two-way ANOVA (****, p <0.0001).

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Infection, Incubation, Flow Cytometry, Reverse Transcription, Quantitative RT-PCR, Transfection, Expressing

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) Cells were collected at 24h after transfection of minigenome or the control plasmid, and half of them were lysed to isolate ribosomes. Top: Total cytoplasmic ribosomes were separated by sucrose density gradient centrifugation, and the absorbance of each fraction was measured at 254nm. Cycloheximide was present in each sample. Lower panel: Protein in half of each fraction’s volume was subjected to TCA precipitation and subsequently utilized for immunoblotting with anti-His and anti-rpS6 antibodies. (B) The remaining half of the cells were extracted for total RNA to detect the mRNA of GFP by quantitative RT-PCR. The results represent the mean ± SD of a representative quantitative RT-PCR experiment conducted in triplicate. (C) Samples from the remaining half of each fraction after ribosome isolation were extracted for RNA and assayed for the distribution of mRNA of GFP in complex with ribosomes by quantitative RT-PCR. Results are the mean ± SD of a representative quantitative RT-PCR experiment performed in duplicate three times. Significance was analyzed by two-way ANOVA. (** means p <0.01, ***means p <0.001). (D) Schematic representation of NP protein deletion mutants. Boxes indicate the protein product of each truncated NP gene, with amino acid positions indicated above the boxes. Straight lines indicate the region of deletion. (E) Residues 122–366 and 366–489 of NP are sufficient for its localization to the ribosome. Multiple c- and n-terminal truncated NPs were expressed in HeLa cells. Cell extracts from transfected cells were subjected to 10–50% sucrose density gradient ultracentrifugation. RNase (100U/mL) was added to the cell lysate to eliminate the impact of varying RNA levels on polyribosome enrichment. Protein in each fraction was subjected to TCA precipitation and subsequently utilized for immunoblotting with anti-His and anti-rpS6 antibodies.

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Transfection, Control, Plasmid Preparation, Gradient Centrifugation, TCA Precipitation, Western Blot, Quantitative RT-PCR, Isolation

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) After HeLa cells were infected with NDVs at 10MOI for 12h, ribosomes were isolated, and the absorbance of each component was measured at A254nm. RNA was extracted from each component, and mRNA of NP (B), HN (C), IFN-α (D), IFN-β (E) and β-actin (F) were detected by quantitative RT-PCR. (G) HeLa cells were infected with NDVs at 1MOI or 10MOI, and protein expression was analyzed at 24h and 36h using relative antibodies. (H) qRT-PCR was performed to detect mRNA expression of IFN-α and IFN-β in HeLa cells infected with NDV at 1MOI and 10MOI at 24h. (I) Expression of IFN-α and IFN-β in cell supernatants at 24h was detected by ELISA. Results are shown as the mean ± SD of three independent experiments. And significance was analyzed by two-way ANOVA (****, p <0.0001).

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Infection, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) Co-immunoprecipitation (Co-IP) of NP protein with endogenous eIF4A1 during NDV infection. HeLa cells were infected with rHerts/33, rI 4 , or mock-infected and subjected to IP using anti-NP protein or anti-eIF4A1 antibodies. Immunoblotting was performed using the indicated antibodies. (B) HeLa cells were transfected with His-tagged HNP or INP and HA-tagged eIF4A1. After 36 hours, HNP’s interaction with eIF4A1 was confirmed through Co-IP using both anti-NP and anti-HA antibodies. (C) GST pull-down assay. Glutathione beads were conjugated with GST or GST-NP fusion protein and incubated with lysate from cells overexpressing eIF4A1. Eluted proteins were subjected to Western Blot, and eIF4A1 presence was detected using anti-HA antibody. GST, GST-HNP, and GST-INP protein expression was confirmed with anti-GST antibody. (D) Redistribution and colocalization of eIF4A1 with HNP protein. HeLa cells were transfected with His-tagged HNP or INP and HA-tagged eIF4A1, fixed at 24 hpi, stained with anti-eIF4A1 and anti-NP antibodies, and visualized using confocal microscopy. (E) Direct interaction between eIF4A1 and HNP confirmed by Bifc assay. Venus luminescence was observed after separate or co-transfection of VC155-eIF4A1, VN173-HNP, and VN173-INP. (F) The C-terminal region 366-489aa of NP is sufficient for heterologous protein association with eIF4A1. Multiple C-terminal and N-terminal truncations of His-tagged NP were expressed in HeLa cells and subjected to IP experiments using anti-eIF4A1 antibody, followed by analysis with specific antibody immunoblotting.

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Infection, Western Blot, Transfection, Pull Down Assay, Incubation, Expressing, Staining, Confocal Microscopy, Bimolecular Fluorescence Complementation Assay, Cotransfection

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A-D) HeLa cells were transfected with either si-NC or si-eIF4A1 for 24 h, followed by infection with rHerts/33 or rI 4 at 1MOI. (A) Cells were harvested at 24 hpi to assess viral protein expression. (B) Cell culture supernatants were collected at 12-hour intervals post-infection until 48 hours, and the virus growth curve was determined by measuring TCID 50 values. (C-D) RNA was extracted at 24 hpi to examine the expression of virus-related mRNA. (E-F) HeLa cells, infected with either rHerts/33 or rI4, were harvested at 6, 12, 18, and 24 hpi for Western Blot analysis using specific antibodies. (G) HeLa cells were pre-treated with the Akt activator SC79 (5 μM) or the Akt inhibitor LY294002 (20 μM) for 2 hours, followed by infection with rHerts/33 or rI 4 . Western Blot analysis was performed at 24 hpi using relevant antibodies. Equal volume DMSO treatment was served as a control. (H) HeLa cells were infected with rI4 and rHerts/33, and were harvested at 24 hpi to assess the mRNA expression of Myc, cyclin B1, and cyclin A2 in each group. The mRNA expression of target genes were normalized to β-actin mRNA and presented as fold induction. (I-J) HeLa cells were transfected with pCAGGS empty vector, pCAGGS-HNP-His, or pCAGGS-INP-His for 24 hours, followed by a 1-hour treatment with either DMSO or the eIF4A1 inhibitor Roc-A (3nM). In (I), the mRNA expression levels of target genes were measured and normalized to β-actin mRNA, presented as fold induction. In (J), Western Blot analysis was performed using relevant antibodies. Results are shown as the mean ± SD of three independent experiments. * p < 0.05 (considered as a significant difference), ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns (not significant) ≥ 0.05 (no significant difference).

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Transfection, Infection, Expressing, Cell Culture, Virus, Western Blot, Control, Plasmid Preparation

Journal: PLOS Pathogens

Article Title: The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency

doi: 10.1371/journal.ppat.1012027

Figure Lengend Snippet: (A) A lentiviral packaging system was used to express NDV-NP protein in HeLa cells, and NP expression was detected by Western blotting. (B) The statistical plot of the number of differentially expressed genes (DEGs) in each group (fc>1, p <0.01). Heat map of (C) HNP DEGs and (D) INP DEGs (fc>1, p <0.01). (E) Heatmap depicting the enrichment of genes dependent on eIF4A1-mediated translation in the ribosome. (F-H) Quantitative RT-PCR to detect the expression of cyclin A2 (F), Myc (G) and cyclin B1 (H). (I) Co-localization of NP protein with puromycin. Cells were transfected with pCAGGS-HNP-His, pCAGGS-INP-His, and control vector, followed by incubation with 208μM emetine at 37°C for 15 min to "freeze" the extended ribosome on the mRNA. This was followed by co-incubation with 10 μg/ml of puromycin (PMY, a tyr-tRNA mimetic) at 37°C for 5 min, and expression of NP and puromycin was observed by confocal microscopy using anti-His and anti-puromycin antibodies. Results are shown as the mean ± SD of three independent experiments. ****means p <0.0001.

Article Snippet: The human epithelial carcinoma cell line HeLa, human non-small cell lung cancer cell line A549, human colon cancer cell line HT29, human colorectal adenocarcinoma cell line HCT116 and the DF1 Chicken Embryo Fibroblast cell line were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Thermo Fisher Scientific, Waltham, MA, USA), in an incubator at 37°C containing 6% CO 2 .

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Incubation, Confocal Microscopy

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Co-IP assay to verify the interaction of TDP-43 with PDI in HEK-293T cells transiently co-transfected with TDP-43 and HA-tagged wild-type PDI or HA-tagged dnPDI treated with 1 mM H 2 O 2 for 1 h. Anti-TDP-43 antibody-binding beads were used for co-IP experiments and then detected by western blotting using anti-HA, anti-TDP-43, and anti-β-actin antibodies. ( B and C ) Schemes of TDP-43-PDI constructs used in BiFC assay: TDP-43-EGFP 1-172 fusion protein (B) and signal peptide-HA-EGFP 155-238 -a-b-b’-x-a’-c fusion protein (C). ( D ) Specific interaction between TDP-43 and PDI in living cells was detected by BiFC. HEK-293T cells transiently expressing both TDP-43-EGFP 1-172 and EGFP 155-238 -wild-type PDI or EGFP 155-238 -dnPDI constructs were treated with 1 mM H 2 O 2 for 1 h and cultured for 1 day. Shown are nuclei stained with Hoechst 33342 (blue). EGFP (green) was observed in (D). ( E ) TDP-43 selectively recruits PDI into its phase-separated condensate. Fluorescence images of in vitro phase-separated droplets (red; Merge: yellow) of 10 μM TAMRA-labeled bacterial-purified TDP-43 incubated with Tris-HCl buffer (pH 8.0) containing 10 μM FITC-labeled bacterial-purified wild-type PDI, dnPDI, or SNO-PDI (green) and 10% (w/v) PEG 3350 on ice, and FITC-labeled BSA as a control. ( F ) Regulation of TDP-43 LLPS by PDI. Fluorescence images of 2.5, 5, 7.5, or 10 μM recombinant TDP-43 labeled by TAMRA (red) and incubated with Tris-HCl buffer containing wild-type PDI, dnPDI, or SNO-PDI at 10 μM and 10% PEG 3350 on ice. Scale bars, 10 μm. ( G to J ) The dependence of turbidity changes for LLPS of TDP-43 (G), TDP-43 + 10 μM wild-type PDI (H), TDP-43 + 10 μM dnPDI (I), or TDP-43 + 10 μM SNO-PDI (J) on the concentration of TDP-43 ([TDP-43]) was expressed as mean ± SD (with error bars) of values obtained in three independent experiments. The turbidity of TDP-43 condensates was measured at 600 nm at 25 °C. Representative calculation based on turbidity measurements to determine saturation concentrations of TDP-43 or TDP-43 + dnPDI (open circle) and TDP-43 + wild-type PDI or TDP-43 + SNO-PDI (open square). The orange and the red lines are drawn through data points indicating the absence of LLPS, whereas the cyan and the blue lines are drawn through data points in which robust LLPS of TDP-43 occurs. The concentration of protein at which these two lines intersect is an estimation of the saturation concentration. ( K and L ) Saturation concentration of TDP-43 (K) and turbidity of TDP-43 condensates (L) (open red circles shown in scatter plots) were expressed as the mean ± SD (with error bars) of values obtained in three biologically independent experiments. TDP-43 + wild-type PDI, P = 0.000002 (K) and 0.00012 (L); TDP-43 + dnPDI, P = 0.0545 or 0.00000068 (K) and 1.0 or 0.00012 (L); and TDP-43 + SNO-PDI, P = 0.0299 or 0.0000000092 (K) and 0.0161 or 0.0029 (L). Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 relative to control (the saturation concentration for TDP-43 or TDP-43 + wild-type PDI). n.s., no significance.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Transfection, Binding Assay, Western Blot, Construct, Bimolecular Fluorescence Complementation Assay, Expressing, Cell Culture, Staining, Fluorescence, In Vitro, Labeling, Purification, Incubation, Control, Recombinant, Concentration Assay

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for endogenous TDP-43 in the cytoplasmic and nuclear protein fractions and the corresponding cell lysates from N2a cells transiently expressing HA-tagged wild-type PDI or HA-tagged dnPDI treated with 1 mM H 2 O 2 for 1 h. β-actin and histone H3 served as markers for cytosol and nuclear fractions, respectively, and empty vector pcDNA3.1 as a control. ( B and C ) The normalized amount of endogenous TDP-43 in cytoplasm (B) and nucleus (C) of the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.000086 (B) and 0.00048 (C); and dnPDI + H 2 O 2 , P = 0.535 or 0.00026 (B) and 0.0104 or 0.0081 (C). Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( D ) Immunofluorescence imaging of N2a cells transiently expressing both EGFP-TDP-43 and empty vector pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibody against PDI (red) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (D). Scale bar, 10 μm.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for endogenous TDP-43 in the cytoplasmic and nuclear protein fractions and the corresponding cell lysates from HEK-293T cells transiently expressing HA-tagged wild-type PDI or HA-tagged dnPDI treated with 1 mM H 2 O 2 for 1 h. β-actin and histone H3 served as markers for cytosol and nuclear fractions, respectively, and empty vector pcDNA3.1 as a control. ( B and C ) The normalized amount of endogenous TDP-43 in cytoplasm (B) and nucleus (C) of the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00049 (B) and 0.00099 (C); and dnPDI + H 2 O 2 , P = 0.741 or 0.00023 (B) and 0.200 or 0.0013 (C). Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; and *** P < 0.001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( D ) Immunofluorescence imaging of HEK-293T cells transiently expressing both EGFP-TDP-43 and empty vector pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibody against PDI (red) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (D). Scale bar, 10 μm.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Co-IP assay to verify the interaction of endogenous TDP-43 with endogenous G3BP1 in N2a cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. Anti-TDP-43 antibody-binding beads were used for co-IP experiments and then detected by western blotting using anti-G3BP1, anti-TDP-43, anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of immunoprecipitated G3BP1 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00031; and dnPDI + H 2 O 2 , P = 0.270 or 0.00073. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; and *** P < 0.001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. (C ) Immunofluorescence imaging of N2a cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and G3BP1 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (C). White arrows indicate colocalization of TDP-43 and endogenous G3BP1 in stress granules. Scale bar, 10 μm.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, Binding Assay, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for phosphorylated endogenous TDP-43 (pTDP-43) in N2a cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. The cell lysates from the aforementioned cells were probed by anti-TDP-43, anti-pTDP-43 (pSer409/410), anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of pTDP-43 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00083; and dnPDI + H 2 O 2 , P = 0.211 or 0.00026. ( C ) Immunofluorescence imaging of N2a cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and pTDP-43 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in ( C ). Scale bar, 10 μm. ( D ) Western blot for endogenous TDP-43 in the sarkosyl-insoluble pellets (including insoluble full-length TDP-43 and insoluble C-terminal TDP-43 fragment of 35 kDa) and the corresponding cell lysates from the same N2a cells as in (A). β-actin served as the protein loading control. ( E ) The normalized amount of TDP-43 aggregates in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.000091; and dnPDI + H 2 O 2 , P = 0.0109 or 0.00026. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; *** P < 0.001; and **** P < 0.0001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( F to I ) Immunogold electron microscopy of TDP-43 aggregates purified from the same N2a cells transiently expressing pcDNA3.1 (F and G), wild-type PDI (H), or dnPDI (I) treated without (F) or with (G−I) 1 mM H 2 O 2 as in (A), and labeled by gold particles conjugated with anti-TDP-43 antibody. Scale bar, 20 nm.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Immunofluorescence, Imaging, Control, Staining, Electron Microscopy, Purification, Labeling

Journal: bioRxiv

Article Title: Protein disulfide isomerase disassembles stress granules and blocks cytoplasmic aggregation of TDP-43 in ALS

doi: 10.1101/2024.03.16.585334

Figure Lengend Snippet: ( A ) Western blot for phosphorylated endogenous TDP-43 (pTDP-43) in HEK-293T cells transiently expressing empty vector pcDNA3.1, transiently expressing pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h, transiently expressing wild-type PDI treated with 1 mM H 2 O 2 for 1 h, and transiently expressing dnPDI also treated with 1 mM H 2 O 2 for 1 h. The cell lysates from the aforementioned cells were probed by anti-TDP-43, anti-pTDP-43, anti-HA, and anti-β-actin antibodies. ( B ) The normalized amount of pTDP-43 in the aforementioned cells (open red circles shown in scatter plots) was expressed as the mean ± SD (with error bars) of values obtained in three independent experiments. Wild-type PDI + H 2 O 2 , P = 0.00027; and dnPDI + H 2 O 2 , P = 0.977 or 0.00095. Statistical analyses were performed using two-sided Student’s t -test. Values of P < 0.05 indicate statistically significant differences. The following notation is used throughout: * P < 0.05; ** P < 0.01; and *** P < 0.001 relative to control (pcDNA3.1 + H 2 O 2 or wild-type PDI + H 2 O 2 ). n.s., no significance. ( C ) Immunofluorescence imaging of HEK-293T cells transiently expressing both EGFP-TDP-43 and pcDNA3.1 (control), transiently expressing both EGFP-TDP-43 and pcDNA3.1 treated with 1 mM H 2 O 2 for 1 h (control + H 2 O 2 ), transiently expressing both EGFP-TDP-43 and wild-type PDI treated with 1 mM H 2 O 2 for 1 h (wild-type PDI + H 2 O 2 ), and transiently expressing both EGFP-TDP-43 and dnPDI also treated with 1 mM H 2 O 2 for 1 h (dnPDI + H 2 O 2 ), using antibodies against PDI (red) and pTDP-43 (magenta) and staining with DAPI (blue). EGFP-TDP-43 (green) was observed in (C). Scale bar, 10 μm.

Article Snippet: The cell lysates were boiled in SDS-PAGE loading buffer for 10 min and then subjected to SDS-PAGE and probed with the following specific antibodies: mouse anti-TDP-43 monoclonal antibody (Abcam, ab57105,1:1000), rabbit anti-TDP-43 polyclonal antibody (Proteintech, 10782-2-AP, 1:2000), rabbit anti-HA tag polyclonal antibody-ChIP Grade (Abcam, ab9110, 1:1000), mouse anti-HA tag monoclonal antibody (Proteintech, 66006-2-Ig, 1:5000), rabbit anti-Histone-H3 polyclonal antibody (Proteintech, 17168-1-AP, 1:1000), mouse anti-G3BP1 monoclonal antibody (Proteintech, 66486-1-Ig, 1:5000), rabbit anti-phospho-TDP-43 (pS409/410) polyclonal antibody (Proteintech, 22309-1-AP, 1:1000), mouse anti-β-actin monoclonal antibody (Proteintech, 66009-1-Ig, 1:5000), HRP-labeled goat anti-rabbit IgG (H+L) (Beyotime, A0208, 1:5000), and HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, A0216, 1:5000).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: The pro-oncogenic adaptor CIN85 inhibits hypoxia-inducible factor prolyl hydroxylase-2

doi: 10.1101/466946

Figure Lengend Snippet: CIN85 interacts with PHD2. (A) Endogenous PHD2 was immunoprecipitated (IP) with CIN85 antibody from MDA-MB-231, BT-549, and Hs 578T cells. Blots from the input were probed with PHD1, −2, −3, CIN85, HIF-1α, and HIF-2α antibody. (B) Western blot (WB) analysis of immunoprecipitates (IP) and WCE from HEK-293 cells, expressing V5-tagged PHD1, PHD2, or PHD3 and Myc-tagged CIN85. Blots from IPs were probed with V5-tag antibody, the input was probed with V5-tag, Myc-tag and α-tubulin antibodies. (C) Schematic presentation of BiFC assay constructs. The constructs CMV-CIN85-YN and CMV-PHD2-YC allow expression of CIN85 and PHD2 as fusion proteins with the N-terminal or the C-terminal non-fluorescent parts of YFP (-YN and -YC) under the control of the CMV promoter, respectively. Note that CMV-YN and CMV-YC protein parts are non-fluorescent and non-interacting; however, interacting proteins such as CIN85 and PHD2 are able to reconstitute fluorescent YFP. (D) Visualization of the BiFC signal by confocal microscopy in BT-549 cells expressing CMV-CIN85-YN+CMV-PHD2-YC. No signal was detected upon expression of CMV-YN+CMV-YC. Scale bar 20 μm. (E) The surface plasmon resonance sensorgram of CIN85 binding to immobilized PHD2. Binding was assessed upon injection of a concentration series of CIN85 over immobilized PHD2. The CIN85 concentrations were 0, 10, 21, 42, 84, 168, 337, 675, 1350, 2700 nM (from bottom to top). (F) The fitted curve for different concentrations of CIN85 binding to immobilized PHD2 using the ‘Affinity’ model in the Biacore T200 evaluation software.

Article Snippet: Human embryonic kidney 293 (HEK-293, # CRL-1573), human breast carcinoma cell lines (MDA-MB-231 (#HTB-26), Hs578T (#HTB-126) and BT-549 (#HTB-122) were purchased from ATCC.

Techniques: Immunoprecipitation, Western Blot, Expressing, Bimolecular Fluorescence Complementation Assay, Construct, Control, Confocal Microscopy, SPR Assay, Binding Assay, Injection, Concentration Assay, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Panel A: Aliquots were separated on an 8% SDS-PAGE and then immunoblotted for the indicated proteins. Gels were cropped to focus only on the high molecular weight SDS-resistant bands. Samples also were blotted for factor B to verify cleavage as an indicator of complement activation. The 75 kDa β-chain of C3 was utilized as a loading control. Panel B: C5a ELISA of complement activation in serum. Panel C: C5a ELISA of complement activation in citrated plasma.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Clinical Proteomics, Activation Assay, SDS Page, High Molecular Weight, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Generation of Multiple Fluid-Phase C3b:Plasma Protein Complexes During Complement Activation. Possible Implications in C3 Glomerulopathies

doi: 10.4049/jimmunol.1302288

Figure Lengend Snippet: Panel A: The amount of C5a generated in reconstituted C3-depleted serum samples measured by ELISA. Antibody coated sheep erythrocytes were treated with either C3-depleted serum alone or C3-depleted serum reconstituted with 1.3 mg/ml native C3, hydroxylamine treated C3 (C3-NH2OH) or C3b and incubated at 37°C for 1 hour. The supernatant was removed and C5a levels quantified by C5a ELISA. Panel B: C3-depleted serum alone or reconstituted with native C3 or hydroxylamine treated C3 (C3-NH2OH), both at 1.3 mg/ml, were activated with CVF (416 U/ml) plus 0.4 mM Mg2+ for 15 minutes at 37°C. CVF activated normal human serum was included as a positive control. Aliquots were separated using 8% SDS-PAGE and immunoblotted for DBP, α1PI, α1AG, factor B and C3. Panel C: Pooled normal human serum was sham-treated with PBS (lane 1) or complement was activated by incubating serum at 37°C using either 416 U/ml CVF (lane 2), 10 mg/ml zymosan A (lane 3), 0.5 mg/300 μl heat-aggregated human IgG (lane 4). As a control to inhibit complement activation, 10 mM EDTA was added to serum prior to addition of CVF (lane 5). Serum aliquots were separated on an 8% SDS-PAGE and then immunoblotted for C3. Panel D: Pooled normal human serum or citrated plasma were activated at 37°C with 416 U/ml CVF for the indicated times. In addition to CVF, 2 mM Mg2+ was added to plasma to overcome the chelation effects of sodium citrate. At each time-point, activation was stopped by placing the sample on ice. Aliquots were separated on an 8% SDS-PAGE and then immunoblotted C3.

Article Snippet: Briefly, Maxi-sorb 96 well plates were coated with 500 ng of mouse monoclonal anti-human C5a (clone 295003; R&D Systems, Minneapolis, MN) capture antibody at 4°C overnight.

Techniques: Generated, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, SDS Page, Control, Activation Assay, Clinical Proteomics

Journal: Oncology Reports

Article Title: Anti-EpCAM monoclonal antibody exerts antitumor activity against oral squamous cell carcinomas

doi: 10.3892/or.2020.7808

Figure Lengend Snippet: CDC activity of an anti-EpCAM mAb in OSCC cells. CDC activity of EpMab-16, control mouse IgG 2a , and control PBS in (A) SAS and (B) HSC-2 cells. Values represent the mean ± SEM. **P<0.01 (determined by ANOVA and Tukey's multiple comparisons tests). CDC, complement-dependent cytotoxicity; EpCAM, epithelial cell adhesion molecule; mAb, monoclonal antibody; OSCC, oral squamous cell carcinoma; PBS, phosphate-buffered saline; SEM, standard error of the mean; n.s., not significant.

Article Snippet: Target cells were plated in 96-well plates, at 2×10 4 cells/well, and 10% rabbit complement (Low-Tox-M rabbit complement; Cedarlane Laboratories), 100 μg/ml of anti-EpCAM antibodies, or control IgG (mouse IgG 2a ) was added to each well.

Techniques: Activity Assay, Control, Saline