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: 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: 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: 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: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 3. Clusterin in human pituitary adenomas. Confocal image of A) human pituitary adenoma and non-tumorous pituitary tissue specimens showing clusterin (green) expressed exclusively in the cytoplasm; B) Co-localization of clusterin with GH, PRL and aGSU in respective human pituitary adenoma specimens (clusterin green, respective hormones red). doi:10.1371/journal.pone.0017924.g003

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques:

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 4. Pituitary proliferation, DNA damage and senescence markers in the aGSU.PTTG pituitary gland. A) In vivo BrdU incorporation. Mice were injected with BrdU (50 mg/g BW), and pituitary sections stained for BrdU. One thousand cells/section, 3 sections/animal, n = 3 animals/ genotype were analyzed. *, p,0.05; Western blot analysis of B) proliferation markers; C) DNA damage, DNA repair and p53-dependent senescence markers, and D) oncogene-induced senescence markers; E) Confocal image showing immunofluorescent cytoplasmic clusterin, and intranuclear p15 and p16 expression (green) in WT and in pre-tumorous aGSU.PTTG pituitary glands, and in aGSU.PTTG pituitary adenomas; F) Pituitary SA-b- galactosidase enzymatic activity (blue) in WT and in pre-tumorous aGSU.PTTG pituitary gland. Three pituitary cryosections/animal were analyzed from 3 animals/genotype, and a representative image shown. Western blots here and elsewhere were repeated 3 times with similar results and representative blots shown. doi:10.1371/journal.pone.0017924.g004

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: In Vivo, BrdU Incorporation Assay, Injection, Staining, Western Blot, Expressing, Activity Assay

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 6. C/EBPs induce clusterin. A) C/EBPb is up-regulated in the aGSU.PTTG pituitary. Confocal image showing C/EBPb co-localization with aGSU-positive, GH-positive and PRL-positive cells in WT and pre-tumorous aGSU.PTTG pituitary glands. (Hormones-green, cytoplsmic, C/EBPb–red, intranuclear); B) Western blot analysis of C/EBPb and d isoforms induced in LbT2 cells stably transfected with mPttg; C) Effects of C/EBPs on the clusterin promoter in LbT2 and aT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and 800 ng murine pCDNA3-C/EBPa, b or d. The ratio of luciferase to co-trasfected b-galactosidase control reporter vector was normalized to pCDNA3- null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown.*, p,0.05, **,p,0.01; D) Western blot analysis of clusterin expression in gonadotroph-derived aT3 cells 24 hours after transfection with pCDNA3-C/EBPb or E) pCDNA3-C/EBPd; F) Western blot analysis of clusterin expression in LbT2 mPttg cells 48 hours after simultaneous transfection with siC/EBPb and siC/EBPd (3 nM each). Two different combinations of siRNAs were used. doi:10.1371/journal.pone.0017924.g006

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Luciferase, Control, Expressing, Derivative Assay

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 7. Clusterin restrains pituitary cell proliferation by inducing Cdk inhibitors. Western blot analysis of Cdk inhibitors and proliferation markers A) in LbT2 cells, B) in aT3 cells 48 h after transfection with mClu; C) Confocal images of immunofluoprescence of histone H3 methylation on lysine 9 (H3-K9M) (red) in vector and Clu-expressing aT3 cells 48 hours after transfection; D) Quantification of positive H3-K9M foci. Cells were fixed, stained with H3-K9M antibody, and one thousand cells/field counted in three randomly chosen visual fields; E) Percentage of BrdU positive cells 48 h after transfection with mClu. Triplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry, *, p,0.05; F) aT3 cells stably overexpressing mClu or vector were synchronized in 0.1% fetal bovine serum for 18 hours, and then cultured in 10% fetal bovine serum. At the indicated times, duplicate samples were pulsed with BrdU for 30 min, analyzed by flow cytometry, and cells in S-phase identified by staining with BrdU antibodies. doi:10.1371/journal.pone.0017924.g007

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Transfection, Methylation, Plasmid Preparation, Expressing, Staining, Flow Cytometry, Stable Transfection, Cell Culture

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 8. Clusterin attenuation promotes proliferation. Western blot analysis of Cdk inhibitors and proliferation markers A) in LbT2 cells, B) in aT3 cells; C) Percentage of BrdU positive cells 48 h after transfection with siClu. D) Upper panel, Western blot confirms p15 down-regulation, Lower panel, Percentage of BrdU positive LbT2 cells 48 h after transfection with sip15. E) Upper panel, Western blot confirms p16 down-regulation, Lower panel, Percentage of BrdU positive LbT2 cells 48 h after transfection with sip16. For BrdU detection, cells were fixed, stained with BrdU antibody and one thousand cells/field in three randomly chosen fields counted. *, p,0.05. doi:10.1371/journal.pone.0017924.g008

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Western Blot, Transfection, Staining

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 9. FOXL2 stimulates the clusterin promoter. A) Effects of FOXL2 on the clusterin promoter in aT3 cells 24 h after transfection. Cells were co-transfected with 200 ng murine pGL3-luc-mClu reporter plasmid and indicated amounts of pcDNA3-His-mFoxl2. The ratio of luciferase to co- trasfected b-galactosidase control reporter vector was normalized to pCDNA3-null expression vector. SEM was calculated from triplicate assays, and experiments repeated three times with similar results. Results of a representative experiment are shown; **,p,0.01; B) Western blot analysis of clusterin expression in aT3 cells 24 hours after transfection with pcDNA3-His-mFoxl2; C) ChiP assay was performed in nuclear fractions derived from aT3 cell lysates. Top, schematic of the approximate location of primers used in the PCR reactions. Enrichment of specific clusterin promoter sequences was obtained with primer Set 2. FOXL2, specific antibody, IgG, nonspecific antibody, PCP, positive control primers. The experiment was repeated twice, and results of a representative assay shown. doi:10.1371/journal.pone.0017924.g009

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Transfection, Plasmid Preparation, Luciferase, Control, Expressing, Western Blot, Derivative Assay, Positive Control

Journal: PloS one

Article Title: Lineage-specific restraint of pituitary gonadotroph cell adenoma growth.

doi: 10.1371/journal.pone.0017924

Figure Lengend Snippet: Figure 10. Proliferation restricting pathways in the pituitary gonadotroph cell lineage. FOXL2 directly activates the clusterin promoter, while Pttg overexpression results in proliferation, DNA damage and stimulation of C/EBPb and d; C/EBPs activate the clusterin promoter. High levels of secretory clusterin trigger expression of Cdk inhibitors p15, p16 and p27, and C/EBPb also cooperates to induce p15. Up-regulated tumor suppressor proteins likely underlie proliferation restraint preventing uncontrolled growth of benign pituitary adenomas of gonadotroph cell origin. doi:10.1371/journal.pone.0017924.g010

Article Snippet: Short hairpin RNA expressing vector targeting murine clusterin shClu-pGFP-V-RS (shClu) were purchased from OriGene( Rockvillle, MD).

Techniques: Over Expression, Expressing

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: 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: 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: 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

Journal: PLoS ONE

Article Title: Deficiency of C5L2 Increases Macrophage Infiltration and Alters Adipose Tissue Function in Mice

doi: 10.1371/journal.pone.0060795

Figure Lengend Snippet: Retroperirenal adipose tissue mRNA gene expression of complement receptors C5L2 (A), C5aR (B) and C3aR (C) analysis of wild-type control (Ctl) (circle and dotted line) and C5L2 −/− (square and solid line) mice on chow diet (white circle or square) or diet induced obesity (DIO) (black circle or square) diet over 6, 12 and 24 weeks. The results are expressed as relative expression (re) compared to 6 weeks chow diet Ctl set as 1. Complement C5a production by primary isolated adipocytes of inguinal (D) and perigonadal (E) adipose tissue in Ctl (white bars) and C5L2 −/− (black bars) mice on chow diet (plain bars) or DIO (hatched bars). Data are presented as mean±SEM (n = 4–10 mice per group) with two-way (2-way) ANOVA for genotype × time between Ctl and C5L2 −/− for each diet with a Bonferroni post-test where * p <0.05 and *** p <0.001 for Ctl vs. C5L2 −/− for the same diet.

Article Snippet: Plasma and adipocyte cultured media were analyzed with mouse Duoset ELISA kits for adiponectin/Acrp30, CCL2/JE (MCP-1), KC/CXCL1 and complement component 5a (C5a) according to commercial assay protocols (R & D systems Inc., Minneapolis, MN, USA).

Techniques: Gene Expression, Control, Expressing, Isolation