Journal: Cell Reports Medicine

Article Title: Single-cell analysis of neoplastic plasma cells identifies myeloma pathobiology mediators and potential targets

doi: 10.1016/j.xcrm.2024.101925

Figure Lengend Snippet:

Article Snippet: IDT® for Illumina DNA/RNA UD Indexes Set A, Tagmentation , Illumina , 20027213.

Techniques: Recombinant, Selection, Cell Isolation, Proliferation Assay, Staining, Enzyme-linked Immunosorbent Assay, Detection Assay, Reporter Assay, RNA Sequencing, Transgenic Assay, Software

Journal: iScience

Article Title: The different impact of drug-resistant Leishmania on the transcription programs activated in neutrophils

doi: 10.1016/j.isci.2024.109773

Figure Lengend Snippet:

Article Snippet: NovaSeq 6000 S1 Reagent Kit v1.5 (100 cycles) , Illumina , Cat#20028319.

Techniques: Control, Recombinant, Isolation, Selection, Staining, Reverse Transcription, Purification, SYBR Green Assay, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cells

Article Title: Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages

doi: 10.3390/cells11101630

Figure Lengend Snippet: ROP7 in THP-1 cells activated NLRP3 inflammasome and induced a hyperactive status in cells. THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h. THP-1 infected with lentivirus as a mock control, THP-1 Mock . ( A ) NLRP3 and ASC were co-precipitated with Flag-HA-ROP7 in THP-1 cells. After expression and differentiation, the cells were harvested for Co-IP using an anti-HA antibody to immunoprecipitated Flag-HA-ROP7 and further analyzed by Western blot using an anti-NLRP3, anti-ASC, and anti-Flag antibody. ( B ) After differentiation, cell medium was renewed and collected periodically to detect IL-1β and TNF-α secretion by ELISA. Data are expressed as mean ± SEM values. * p < 0.05, *** p < 0.001, as compared with THP-1 Mock . ( C ) pro-IL-1β and p17 of THP-1 Mock and THP-1 ROP7 in cell lysate and supernatant (SN) were detected by immunoblotting using anti-IL-1β. In total, 100-ng/mL LPS was used in THP-1 Mock for 4 h as a positive control. ( D ) At 24 h post-induction, the cells were treated with 100-ng/mL LPS for 4 h alone or 10-μM nigericin for 1 h subsequently. Supernatants were collected for ELISA. Data are expressed as mean ± SEM values. ** p < 0.01, *** p < 0.001, as compared with THP-1 Mock . ( E ) After PMA differentiation, 10-μM of Z-YVAD-FMK, Z-VAD-FMK, or MCC950 was added to the cells for 24 h. The IL-1β and TNF-α in cell-free supernatant were detected by ELISA. Data are expressed as mean ± SEM values. *** p < 0.001, as compared with the DMSO group. ( F ) Supernatants that were untreated (24 h after PMA differentiation and medium replacement), LPS-treated (100 ng/mL for 4 h), and nigericin-treated (10 μM for 1 h) were collected for the detection of LDH release. The results were normalized by the maximum enzyme activity in each group. Data are expressed as mean ± SEM values. ns: non-significant, ** p < 0.01.

Article Snippet: Antibodies: anti-IL-1β (NB600-633, Novus Biologicals, Littleton, CO, USA); anti-HA (Ab137838, Abcam, Cambridge, UK); anti-Flag (F1804, Sigma-Aldrich, St Louis, MO, USA); anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA); anti-ASC (AG-25B-0006-C100, Adipogen, San Diego, CA, USA) Reagents: recombinant human IL-1β (IL038, Sigma-Aldrich, St Louis, MO, USA), recombinant human TNF-α (ab9642, Abcam, Cambridge, UK); Z-YVAD-FMK (S8507, Selleck, Shanghai, China); Z-VAD-FMK (S7023, Selleck, Shanghai, China); MCC950 (S8930, Selleck, Shanghai, China); Bay 11-7082 (S2913, Selleck, Shanghai, China); IL-1R antagonist (sc-221747, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Infection, Control, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Activity Assay

Journal: Cells

Article Title: Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages

doi: 10.3390/cells11101630

Figure Lengend Snippet: The NF-κB signaling pathway was activated in THP-1 ROP7 , but its activation was not directly derived from ROP7. THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h ( A ) or with 10-μM BAY11-7082 in THP-1 ROP7 . ( B ) Total RNA was extracted, and the mRNA relative fold changes were determined via RT-qPCR. ( C ) The 293T cells were co-transfected with a dual-luciferase reporter plasmid (ranilla: firefly 1:10) for NF-κB. pcDNA3-HA-ROP7 or empty plasmids (mock). At 48 h post-transfection, luciferase activity was examined, and the values were normalized according to the ratio of firefly luciferase activity and ranilla luciferase activity. In total, 20-ng/mL TNF-α was used as a positive control. ( D ) THP-1 ROP7 cells were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h along with 10-μM Z-YVAD-FMK, Z-VAD-FMK, or MCC950. Total RNA was extracted, and the mRNA relative fold changes were determined via RT-qPCR. ( E ) THP-1 Mock and THP-1 ROP7 were induced by 1-μg/mL doxycycline for 48 h and differentiated under 100-nM PMA for another 48 h. Then, 100-ng/mL LPS for 4 h and 10-μM nigericin for 1 h subsequently were used to activate NLRP3 inflammasomes. In total, 10-μM Z-YVAD-FMK, Z-VAD-FMK, or MCC950 was added with LPS or nigericin. The IL-1β and TNF-α supernatant concentrations were detected by ELISA. Data are expressed as mean ± SEM values. ns: non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, as compared with untreated groups.

Article Snippet: Antibodies: anti-IL-1β (NB600-633, Novus Biologicals, Littleton, CO, USA); anti-HA (Ab137838, Abcam, Cambridge, UK); anti-Flag (F1804, Sigma-Aldrich, St Louis, MO, USA); anti-NLRP3 (AG-20B-0014-C100, Adipogen, San Diego, CA, USA); anti-ASC (AG-25B-0006-C100, Adipogen, San Diego, CA, USA) Reagents: recombinant human IL-1β (IL038, Sigma-Aldrich, St Louis, MO, USA), recombinant human TNF-α (ab9642, Abcam, Cambridge, UK); Z-YVAD-FMK (S8507, Selleck, Shanghai, China); Z-VAD-FMK (S7023, Selleck, Shanghai, China); MCC950 (S8930, Selleck, Shanghai, China); Bay 11-7082 (S2913, Selleck, Shanghai, China); IL-1R antagonist (sc-221747, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Activation Assay, Derivative Assay, Quantitative RT-PCR, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: (A) Schematic representation of full-length E2, E2 661 , and E2 661 variants with deletions of HVR1 (Δ1), HVR2 (Δ2), the igVR (Δ3), or combinations thereof (Δ12, Δ13, Δ23, and Δ123). HVR2 and the igVR were replaced with a GSSG linker. Numbering is done according to the H77c prototype strain. Epitope I, II, and III regions are underlined on the E2 structure and overlap CD81 binding sites, shown in blue, orange, and green. A fourth region (yellow) is also implicated in CD81 interactions. Hypervariable region 1, HVR2, and the igVR are shown in red. The transmembrane domain and the C-terminal stem region are shown in black and gray, respectively, on the full-length E2 schematic. (B) Cartoon drawing of the E2 core domain with its surface overlaid (PDB accession number 4MWF ) . Coloring is according to that described above for panel A. The predicted location of the region spanning residues 411 to 420 (purple) that overlaps epitope I and precedes HVR1 is shown.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Characterization of MAbs. (A) Immunoprecipitation of 35 S-labeled H77c E1E2 from lysates of transfected HEK293T cells using each MAb. Immunoprecipitates were run on reducing SDS-PAGE gels and phosphorimaged. Locations of E2 and multiple glycoforms of E1 are shown on the right. Positions of molecular weight markers (M) (in thousands) are shown on the left. Quantitation was performed by using imageQuant software, and results are the means ± standard deviations of data from three experiments (bottom). The antigens to which the MAbs were raised are indicated above the gel. (B) Analysis of the ability of MAbs to recognize denatured E2 661 . Nickel affinity-purified E2 661 was subjected to reducing SDS-PAGE and transferred onto nitrocellulose. Strips were probed with each MAb, followed by detection with anti-mouse Alexa 680-labeled antibody and infrared analysis (Li-COR Odyssey). The antigens to which the MAbs were raised are indicated above the gel. (C) Overlapping-peptide scan of antibodies reactive to denatured E2 661 . Synthetic 18-mers, overlapping the H77c E2 sequence by 11 amino acids, were used in a direct binding ELISA. Binding to a peptide is shown in gray and represents at least 10 times the background level. (D) Ability of MAbs raised to WT E2 661 to recognize an extended HVR1 peptide of strain H. Antibodies were applied to plates coated with peptide and titrated 0.5 log 10 .

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Immunoprecipitation, Labeling, Transfection, SDS Page, Molecular Weight, Quantitation Assay, Software, Affinity Purification, Sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Direct binding of MAbs to E2 661 with single and multiple variable region deletions. Data shown are the means ± standard deviations of data from at least two independent experiments. Percent binding was calculated relative to the binding observed for WT E2 661 . Nonlinear regression analysis was performed with Prism v 6.0f. Data from two independent analyses of MAb24 reactivity are shown for reproducibility. The amounts of E2 661 containing single and multiple deletions of the variable regions added to plates were equivalent, as indicated by GNA-lectin capture of proteins followed by detection with an antibody to the 6×His epitope tag (anti-His) from two independent analyses performed three times (means ± standard deviations).

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Ability of MAbs to inhibit binding between E2 661 or Δ123 and recombinant MBP-LEL 113–201 . (A) Serial dilutions of antibody were mixed with 50 ng E2 E2 661 or Δ123 and applied to plates coated with purified dimeric MBP-LEL 113–201 . Bound E2 was detected with rabbit anti-His and horseradish peroxidase-labeled goat anti-rabbit IgG. Results shown are the means ± standard deviations of data from at least 2 independent experiments. Data were normalized to the percentage of E2 binding to CD81 in the absence of MAb. Curves were fitted with one-site-specific binding with the Hill slope equation in Prism v 6.0f. (B) Binding of E2 661 proteins containing one or more variable region deletions to solid-phase MBP-LEL 113–201 . The inset graph shows the capture of E2 proteins with GNA-lectin and detection with anti-His antibody and confirms that similar amounts of E2 protein were present in every well. (C) Biosensor analysis of binding of E2 661 and variants containing one or more variable region deletions to dimeric MBP-LEL 113–201 . Four concentrations of each E2 661 protein were flowed over biosensor chips coated with MBP-LEL 113–201 , and the curves generated with 100 μg/ml E2 protein are shown.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay, Recombinant, Purification, Labeling, Generated

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Summary of characteristics of the 18 MAbs raised against E2 661 and Δ123

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Neutralization

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Comparative analysis of the abilities of MAbs to inhibit E2 binding to CD81 and dissociation of E2 from CD81

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: Binding Assay

Journal: Journal of Virology

Article Title: Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region

doi: 10.1128/JVI.02070-15

Figure Lengend Snippet: Ability of MAbs to bind their epitopes on the surface of VLPs. (A) VLPs containing genotype 1a H77c E1E2 glycoproteins were pelleted through a sucrose cushion, subjected to reducing SDS-PAGE, and transferred onto nitrocellulose. Membranes were probed with a mixture of H52 (anti-E2) and A4 (anti-E1) or with IgG obtained from an HIV-positive individual. (B) Binding of anticapsid antibody to VLPs is enhanced by permeabilization with Triton X-100. Capsid protein (anti-CA) was detected with MAb183. No binding was observed by using an irrelevant MAb to a Myc epitope tag (anti-myc) or in the absence of primary antibody (No primary). (C) Ability of MAbs to bind VLPs in a direct binding ELISA. Data shown are the means ± standard deviations of data from two independent experiments. OD, optical density.

Article Snippet: The codon-optimized DNA sequence encoding H77c E2 661 was synthesized (GeneArt, Invitrogen, CA, USA) and contained an N-terminal human trypsin leader sequence and a C-terminal 6×His tag.

Techniques: SDS Page, Binding Assay, Enzyme-linked Immunosorbent Assay