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: 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

Journal: Virology

Article Title: Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness.

doi: 10.1016/j.virol.2017.06.034

Figure Lengend Snippet: Fig. 4. PCF degeneration downregulated RSV-induced IFNGR1 expression. (A) Immunohistochemistry staining. The left four pictures have a magnification of ×100 (bar = 100 µm), and the right four pictures are 400× magnification of the red boxes from the corresponding pictures on the left. (B) Western blot. The results are representative of three independent experiments, with a total of 5–7 mice/group, and all data are presented as the mean ± SEM. (*) P < 0.05, and (***) P < 0.001. Experiments were performed as described in Materials and Methods.

Article Snippet: Incubation with rabbit anti-IFNGR1 primary antibody was conducted overnight at 4 °C (1:100 dilution; Abcam, Cambridge, MA), followed by incubation with goat anti-rabbit secondary antibody (1:200 dilution; Boster, Wuhan, China) for 1 h at 37 °C.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Virology

Article Title: Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness.

doi: 10.1016/j.virol.2017.06.034

Figure Lengend Snippet: Fig. 5. IFNGR1 neutralization alleviated RSV-induced acute airway inflammation and AHR. Experiments were performed as described in Materials and Methods. (A) IFNGR1 neutralization reduced the number of total cells and (B) lymphocytes in BALF. (C) IFNGR1 neutralization alleviated RSV-induced pulmonary inflammatory cells infiltration. (D) IFNGR1 neutralization decreased RSV-induced airway resistance. (E) IFNGR1 neutralization did not affect the BALF IFN-γ levels. The results are representative of three independent experiments, with a total of 5–7 mice/group, and all data are presented as the mean ± SEM. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001. Fig (D): (**) P < 0.01, compared with RSV. Tc: Total cells, Mac: Macrophages, Lym: Lymphocytes, Neu: Neutrophils.

Article Snippet: Incubation with rabbit anti-IFNGR1 primary antibody was conducted overnight at 4 °C (1:100 dilution; Abcam, Cambridge, MA), followed by incubation with goat anti-rabbit secondary antibody (1:200 dilution; Boster, Wuhan, China) for 1 h at 37 °C.

Techniques: Neutralization

Journal: Virology

Article Title: Pulmonary C-fiber degeneration downregulates IFN-γ receptor 1 via IFN-α induction to attenuate RSV-induced airway hyperresponsiveness.

doi: 10.1016/j.virol.2017.06.034

Figure Lengend Snippet: Fig. 8. Exogenous recombinant mouse IFN-α (rIFN-α) downregulated RSV-induced IFNGR1 expression. Immunohistochemistry, qPCR and Western blot were done as described in Materials and Methods. (A) rIFN-α treatment induced STAT1 mRNA expression post-RSV infection. (B) rIFN-α treatment induced STAT1 and pSTAT1 protein expression post-RSV infection. (C) rIFN-α treatment reduced RSV-induced IFNGR1 expression detected by immunohistochemistry staining and (D) by Western blot. Magnification ×400, bar represents 100 µm. The results are representative of three independent experiments with a total of 6–8 mice/group, and all data are presented as the mean ± SEM. (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

Article Snippet: Incubation with rabbit anti-IFNGR1 primary antibody was conducted overnight at 4 °C (1:100 dilution; Abcam, Cambridge, MA), followed by incubation with goat anti-rabbit secondary antibody (1:200 dilution; Boster, Wuhan, China) for 1 h at 37 °C.

Techniques: Recombinant, Expressing, Immunohistochemistry, Western Blot, Infection, Staining

Journal: Frontiers in Immunology

Article Title: VISTA + follicular regulatory T cells modulate the function of effector immune cells: implications for ovarian cancer immune escape

doi: 10.3389/fimmu.2025.1704048

Figure Lengend Snippet: Effects of VISTA + /VISTA − Tfr cells on CD8 + T cell proliferation and cytokine secretion. After co-culture of VISTA + or VISTA − Tfr cells with CD8 + T cells (CD8 + T cells: Tfr cells = 4:1): (A) Representative flow cytometry histogram of CFSE proliferation assay for co-cultured cells. The x-axis represents CFSE fluorescence intensity, and the y-axis represents cell count (n=3). (B) Quantitative analysis of CFSE fluorescence intensity in VISTA − Tfr + CD8 + T cell group versus VISTA + Tfr + CD8 + T cell group. (C) Quantitative analysis of CFSE fluorescence intensity after co-culture of CD8 + T cells with VISTA − Tfr cells overexpressing VISTA (LV-VISTA). (D) Quantitative analysis of CFSE fluorescence intensity after co-culture of CD8 + T cells with VISTA + Tfr cells with VISTA silenced (VISTA-shRNA). (E–L) Levels of IL-2, IFN-γ, perforin (PFP), and granzyme B in culture supernatants of VISTA − Tfr + CD8 + T cell group (E–H) and VISTA + Tfr + CD8 + T cell group (I–L) as detected by ELISA (n=3). The statistical results are expressed as the mean ± SD, * p < 0.05, ** p < 0.01.

Article Snippet: Then, 5 μL each of IL-4-APC (MultiSciences, F11IL403), IFN-γ-PE (MultiSciences, F11IFNG02), and IL-17A-FITC (Elabscience, E-AB-F1173C) antibodies were added, followed by incubation at 4°C in the dark for 30 min.

Techniques: Co-Culture Assay, Flow Cytometry, Proliferation Assay, Cell Culture, Fluorescence, Cell Counting, shRNA, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: VISTA + follicular regulatory T cells modulate the function of effector immune cells: implications for ovarian cancer immune escape

doi: 10.3389/fimmu.2025.1704048

Figure Lengend Snippet: Effects of VISTA + /VISTA − Tfr cells on CD25 − CD4 + T cell differentiation. (A) Flow cytometry scatter plots showing the effects of VISTA + /VISTA − Tfr cells in the co-culture system on the expression of differentiation markers IFN-γ, IL-4, and IL-17A on CD25 − CD4 + T cells (n=3). (B–D) Quantitative bar graphs showing the expression proportions of Th1, Th2, and Th17 cells after co-culture of CD25 − CD4 + T cells with VISTA − Tfr cells or VISTA − Tfr cells overexpressing VISTA (LV-VISTA). (E–G) Quantitative bar graphs showing the expression proportions of Th1, Th2, and Th17 cells after co-culture of CD25 − CD4 + T cells with VISTA + Tfr cells or VISTA + Tfr cells with VISTA silenced (shRNA-VISTA). The statistical results are expressed as the mean ± SD, * p < 0.05, ** p < 0.01, ns, no significant difference.

Article Snippet: Then, 5 μL each of IL-4-APC (MultiSciences, F11IL403), IFN-γ-PE (MultiSciences, F11IFNG02), and IL-17A-FITC (Elabscience, E-AB-F1173C) antibodies were added, followed by incubation at 4°C in the dark for 30 min.

Techniques: Cell Differentiation, Flow Cytometry, Co-Culture Assay, Expressing, shRNA