Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB promotes tumorigenesis and anti-tumor immunity in LUAD. A, qRT-PCR analysis of FARSB mRNA levels in different cell groups (si-NC, control group; si-FARSB, FARSB knockdown group). B, WB analysis of FARSB protein expression in various cell groups. C, CCK-8 assay evaluating the effect of FARSB knockdown on cell viability. D, EdU assay assessing the impact of FARSB knockdown on cell proliferation. E and F, Flow cytometry analysis of the effect of FARSB knockdown on cell apoptosis. G, WB analysis of PD-L1 (an immune checkpoint protein) expression levels. H, CFSE fluorescence staining assessing CD8 + T cell proliferation (CFSE dye dilution measures CD8 + T cell division). I-K, Flow cytometry analysis of IFN-γ (I), GZMB (J), and TNF-α (K) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. N = 3, *** p < 0.001 and **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor-alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Quantitative RT-PCR, Control, Knockdown, Expressing, CCK-8 Assay, EdU Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: FARSB Activates the mTOR signaling pathway, promoting LUAD tumorigenesis and anti-tumor immunity. A, GSEA analysis of enriched pathways associated with differential FARSB expression in LUAD. B, WB analysis of the expression of the pathway-related proteins p-mTOR, mTOR, p-p70S6K, p70S6K, p-4EBP1, and 4EBP1. C, CCK-8 assays evaluating cell viability. D, EdU assays assessing cell proliferation. E, Flow cytometry analysis of cell apoptosis. F, WB analysis of PD-L1 expression levels. G, CFSE fluorescence staining assessing CD8 + T cell proliferation. H-J, Flow cytometry analysis of IFN-γ (H), GZMB (I), and TNF-α (J) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8 + T cell cytotoxic function. n = 3, ** p < 0.01, *** p < 0.001, **** p < 0.0001 denote statistical significance. CFSE = carboxyfluorescein succinimidyl ester; FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = mammalian target of rapamycin; ns = no significant differences; PD-L1 = programmed death-ligand 1; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Western Blot

Journal: Journal of the Chinese Medical Association : JCMA

Article Title: Phenylalanyl-tRNA synthetase subunit beta downregulation by spi1 proto-oncogene modulates lung adenocarcinoma progression and immune microenvironment via mammalian target of rapamycin pathway

doi: 10.1097/JCMA.0000000000001286

Figure Lengend Snippet: SPI1 Downregulates FARSB to modulate the mTOR signaling pathway, thus influencing LUAD progression and anti-tumor immunity. A, qRT-PCR analysis of FARSB and SPI1 mRNA levels in different cell groups. B, WB analysis of FARSB and SPI1 protein expression. C and D, WB analysis of mTOR signaling pathway-related proteins. E, CCK-8 assays evaluating cell viability. F, EdU assays assessing cell proliferation. G, Flow cytometry analysis of cell apoptosis. H, WB analysis of PD-L1 expression levels. I: CFSE fluorescence staining assessing CD8 + T cell proliferation. J-L: Flow cytometry analysis of IFN-γ (J), GZMB (K), and TNF-α (L) expressions in tumor-infiltrating CD8 + T cells; these cytokines reflect CD8+ T cell cytotoxic function. n = 3, *** p < 0.001, **** p < 0.0001 denote statistical significance. FARSB = phenylalanyl-tRNA synthetase subunit beta; GZMB = Granzyme B; IFN-γ = interferon-gamma; LUAD = lung adenocarcinoma; mTOR = Mammalian target of rapamycin; ns = no significant differences or significant correlation.; PD-L1 = programmed death-ligand 1; qRT-PCR = quantitative reverse transcription-polymerase chain reaction; TNF-α = tumor necrosis factor alpha; WB = western blot.

Article Snippet: Antibodies included: PE Anti-Human interferon-gamma (IFN-γ) Antibody (E-AB-F1196, Elabscience, Wuhan, China), Granzyme B (GZMB) Monoclonal Antibody (GB11, APC, Thermo Fisher), and tumor necrosis factor-alpha (TNF-α) Monoclonal Antibody (MAb11, APC, 17-7349-82, Thermo Fisher).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Flow Cytometry, Fluorescence, Staining, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Biomedicines

Article Title: Isolation of Functional SARS-CoV-2 Antigen-Specific T-Cells with Specific Viral Cytotoxic Activity for Adoptive Therapy of COVID-19

doi: 10.3390/biomedicines10030630

Figure Lengend Snippet: List of fluorochrome-labeled antibodies used in flow cytometry studies. FITC, fluorescein isothiocyanate; PE, phycoerythrin; PE/Cy7, tandem comprising phycoerythrin and cyanine 7; APC, allophycocyanin; APC/Cy7, tandem comprising allophycocyanin and cyanine 7.

Article Snippet: INF-γ , 45-15 , PE , Miltenyi Biotec , 130-113-493.

Techniques: Flow Cytometry

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Nucleotide sequences of the primers used for real-time PCR

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques: Sequencing

Journal: AIDS Research and Therapy

Article Title: The dynamic changes of interferon lambdas related genes and proteins in JAK/STAT pathway in both acute and chronic HIV-1 infected patients

doi: 10.1186/s12981-017-0158-7

Figure Lengend Snippet: Correlation between the CD4 + T cells and mRNA levels of IFN-alpha receptor ( a ), IFN-gamma receptor ( c ), and IFN-lambdas receptor ( e ). Correlation between the viral loads and mRNA levels of IFN-alpha receptor ( b ), IFN-gamma receptor ( d ), and IFN-lambdas receptor ( f ). The results were performed Spearman’s rank correlation, where coefficients “r” and corresponding p values are indicated on each panel

Article Snippet: Antibodies (Abs) used in this study were as follows: mouse phycoerythin (PE)-conjugated anti-human IFN-alpha/beta R2 Ab (clone MMHAR-2, R&D Systems), mouse PE-conjugated anti-human IFN-lambdas R1 Ab (clone 601106, R&D Systems), mouse PE-conjugated anti-human IFN-gamma Receptor 1 (clone GIR-208, Thermo Fisher Scientific), Mouse eFluor ® 450-conjugated anti-human STAT1 (KIKSI0803, Thermo Fisher Scientific), Rabbit FITC anti-Human STAT2 (Thermo Fisher Scientific), Mouse PE-Cyanine7-conjugated anti-human STAT3 (clone LUVNKLA, Thermo Fisher Scientific), mouse APC-conjugated anti-human STAT4 (clone 4LURPLE, Thermo Fisher Scientific), mouse FITC-conjugated anti-human STAT5 (clone SRBCZX, Thermo Fisher Scientific), mouse PerCP-eFluor ® 710-conjugated anti-human STAT6 (clone CHI2S4N, Thermo Fisher Scientific).

Techniques:

Journal: bioRxiv

Article Title: High-resolution low-cost LCD 3D printing of microfluidics

doi: 10.1101/2023.12.31.573772

Figure Lengend Snippet: (a) ELISA-on-a-chip designed for LCD 3D printing with structurally encoded sequential delivery of reagents to autonomously perform an assay and coupled with a built-in chip-to-assay connection. (b) ELISA workflow showing the sandwich immunoassay designed for the detection of IFN-γ by sequentially delivering assay reagents and wash buffer to a nitrocellulose membrane pre-spotted with anti-human IFN-γ capture antibody; (1) sample containing IFN-γ, (2) biotinylated anti-human IFN-γ detection antibody, (3) streptavidin-conjugated enzyme pHRP, and (4) enzyme substrate in the presence of hydrogen peroxide to generate the colorimetric readout. (c) Autonomous CC workflow for on-chip reagent aliquoting by metering the correct reagent volumes and drainage of the excess, followed by (d) MCR-based sequential delivery of assay reagents with wash steps in between. Arrows show the direction of flow. Scale bar = 5 mm. (e) Binding curve of the on-chip assay for the detection of IFN-γ with a limit of detection of 12 pg mL -1 (CV: 6.8%) across triplicate chips for each tested concentration point; line shows a 4-point logistic fit.

Article Snippet: Purified mouse monoclonal IgG anti-human interferon-γ capture antibody (Cat. #MAB2852, lot #FIO1022021, R&D Systems, Minneapolis, Minnesota, United States), biotinylated affinity purified goat IgG anti-human interferon-γ detection antibody (Cat. #BAF285, lot #ZX2721071, R&D Systems, Minneapolis, Minnesota, United States), recombinant human interferon-γ protein (Cat. #285-IF, lot #RAX2422031, R&D Systems, Minneapolis, Minnesota, United States), Pierce streptavidin poly-horseradish peroxidase (pHRP) (Cat. #21140, lot #XJ360080, Thermo Fisher Scientific, Waltham, Massachusetts, United States), SIGMAFAST 3,3’-Diaminobenzidine tablets (Cat. #D4293, lot #SLCG5357, Sigma-Aldrich, Oakville, Ontario, Canada), bovine serum albumin (BSA) (Cat. #001-000-162, lot #162191, Jackson ImmunoResearch Labs, West Grove, Pennsylvania, United States), BSA-biotin (Cat. #A8549, Sigma-Aldrich, Oakville, Ontario, Canada), Tween 20 (Cat. #P7949, lot #SLBX0835, Sigma-Aldrich, Oakville, Ontario, Canada).

Techniques: Enzyme-linked Immunosorbent Assay, Membrane, Binding Assay, Concentration Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Antigenic stimulation of human splenic mononuclear cells mimics the T CD4 + response of GC reaction (A) Splenic mononuclear cells (splenocytes) were stimulated with CytoStim and cultured for 3 days in the presence of cytokines (IL-7, IL-12, activin) (A). CXCR5 and PD-1 expressions among CD4+ T cells were assessed. Representative flow plots showing CXCR5 and PD-1 expression on CD4 + T cells from ex vivo splenocytes and splenocytes cultured for 3, 5, and 10 days (left) and the percentage of Tfh among CD4+ T cells (right). (B) Representative flow plots showing IL-21 production by Tfh (left) and the percentage of IL-21-positive cells among Tfh (right). (C) Representative histogram showing ICOS expression among Tfh (left) and relative expression of ICOS among Tfh (right). (D) Gating strategy allowing the identification of PD-1 neg Tfh, non-GC Tfh, and GC Tfh among total CXCR5 + PD-1 + cells ex vivo or after 3 days of stimulation of splenocytes or PBMCs in polarizing cytokines. (E) Percentage of total CXCR5 + PD-1 + cells including PD-1 neg Tfh, non-GC Tfh, and GC Tfh among CD4 + T cells ex vivo or after 3 days of culture using splenocytes or PBMCs (n = 7–14). (F and G) Mean fluorescence intensity of PD-1 (F) and CXCR5 (G) expression on ex vivo GC Tfh and GC Tfh D3 splenic cells. (H) Representative flow plots showing IL-21 and IFNγ production by non-GC Tfh D3 and GC Tfh D3 cells 3 days after splenocyte stimulation. (I) Percentage of IL-21- and/or IFNγ-positive cells among non-GC Tfh D3 cells and GC Tfh D3 . (J) Gating strategy for analysis of Bcl6 expression in CD4 + T cells and histograms showing Bcl6 mean fluorescence intensity for GC Tfh D3 , non-GC Tfh D3 , and CXCR5 − PD-1 - CD4 + T cell subsets (n = 14). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005.

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Cell Culture, Expressing, Ex Vivo, Fluorescence

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software