Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Treatment of BH3-mimetic drugs in HCC1195 and HCC1438 cells. (a-c) Cytosol and mitochondrial proteins were extracted from HCC1195, HCC1438, CD3 + T, pulmonary fibroblast cells, and expression of Bcl-2 or Bcl-xL was determined using western blotting. Beta-tubulin was used as a positive marker for the cytosol, while COX IV was used as a positive marker for the mitochondrial fraction. (d) Cell death was measured following treatment with 3 nM ABT199, 19 nM A115463, or 15 µM ABT737 for 1 h, or incubation with 200 pg/µL granzyme B and 100 pg/µL perforin for 2 h. (e, f) The cells were fixed up to 5 h, and apoptosis were determined using ELISA kit. (g) Structures of ABT199 and ABT199-BODIPY. (h) Following treatment of synthetic ABT199-BODIPY, refractive index images of HCC1195 were enlarged at 28, 30, or 32 min, and ABT199-BODIPY were colored in green. (i) Quantified mean fluorescent intensity of the BODIPY signal in cells was measured at 512 nm. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from cytosol fraction, P < 0.05. # Significantly different from treatment of ABT199-BODIPY at 0 min, P < 0.05. COX IV: cytochrome c oxidase subunit IV; BH3: Bcl-2 homology 3; Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; ELISA: enzyme-linked immunoassay; BODIPY: boron dipyrromethene.

Article Snippet: The cells were treated with ABT199 (3 nM, 6960, Tocris, UK), A115463 (19 nM, S7800, Selleck Chemicals, USA), or ABT737 (15 µM, 6835, Tocris, UK) for 1 h. Apoptosis was induced following combinations of recombinant perforin (100 pg/µL, ENZ-PRT313-0010, Enzo Life Science, USA) and granzyme B (200 pg/µL, 10345-H08H, Sinobiological, China).

Techniques: Expressing, Western Blot, Marker, Incubation, Enzyme-linked Immunosorbent Assay, Refractive Index

Journal: Frontiers in Immunology

Article Title: Targeting HIV-1 reservoirs in T cell subsets

doi: 10.3389/fimmu.2023.1087923

Figure Lengend Snippet: Induction of cell death in HIV-1-infected naïve, central memory and effector memory T cells. (A) CD4 + T cells were purified from human PBMCs using anti-CD4-MACS beads (Miltenyi Biotec), and naïve, central memory (CMT) and effector memory T cells (EMT) were sorted by flow cytometry. Sorted T cell subsets were infected with HIV-1 (NL4-3, 1 MOI). Latent infection was then established by culturing with CCL-19 for 4 days, followed by stimulation with IDB for 24 h. Intracellular staining of HIV-1 p24 was determined by flow cytometry. (B, C) The cells with or with latent HIV-1 infection as in (A) were treated for 48 h with IDB (0.1 μM), ABT-263 (0.2 μM) and SAR405 (2 μM) as indicated. The cells were then stained with DEVD-FITC and APC-Annexin V, followed by intracellular staining with PE-anti-HIV p24. Total cell death for cells (B) and the remaining viable HIV-1 p24 + cells negative for staining by APC-annexin V and DEVD-FITC (C) were calculated. Data represent biological replicates and are presented as mean ± SD. P values was determined by unpaired, two-tailed t test in (A) , or by one-way ANOVA with unpaired, two-tailed t -test in (B) and (C) (Control vs. different treatments). ** P <0.01.

Article Snippet: ABT-263 (0.2 μM, Adooq Bioscience) and SAR405 (2 μM, MedChemExpress) were added as indicated.

Techniques: Infection, Purification, Flow Cytometry, Staining, Two Tailed Test, Control

Journal: Frontiers in Immunology

Article Title: Targeting HIV-1 reservoirs in T cell subsets

doi: 10.3389/fimmu.2023.1087923

Figure Lengend Snippet: Specificity in the killing of HIV-1-infected naïve T cells, CMT and EMT, but not uninfected bystander cells. (A, B) HIV-1 latent infection was established in naïve T cells, CMT and EMT as in Figure 4 , and mixed with CellTrace Violet-labeled uninfected counterparts. Cells were cultured with SECH components (IDB, ABT-263 and SAR405) in the presence of BMS-626529 as indicated for 48 h. The cells were then stained with DEVD-FITC and APC-Annexin V, and analyzed by flow cytometry (A) . Percentages of DEVD-FITC + Annexin V + cells were quantified (B) . Data represent biologically replicates and are presented as mean ± SD. Comparison with uninfected cells: ** P <0.01. (C) Viral reactivation with LRAs can induce the expression of HIV-1 components that trigger apoptosis. However, LRAs simultaneously upregulate pro-survival autophagy and anti-apoptotic molecules that counteract viral product-induced cell death, resulting in incomplete killing of HIV-1-infected cells. Inhibiting pro-survival autophagy and anti-apoptotic molecules allows viral products to induce high levels of cell death after treatments with LRAs. In uninfected bystanders, upregulation of pro-survival autophagy and anti-apoptotic molecules by IDB would confer protection against cell death.

Article Snippet: ABT-263 (0.2 μM, Adooq Bioscience) and SAR405 (2 μM, MedChemExpress) were added as indicated.

Techniques: Infection, Labeling, Cell Culture, Staining, Flow Cytometry, Comparison, Expressing

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

Article Title: Disease Risk-Associated Genetic Variants in STAT1 and STAT4 Function in a Complementary Manner to Increase Pattern-Recognition Receptor–Induced Outcomes in Human Macrophages

doi: 10.4049/jimmunol.1901112

Figure Lengend Snippet: JAK1 and TYK2 regulate TLR4-induced cytokines in a manner distinct to STAT1 and STAT4. (A) MDMs were transfected with scrambled, STAT1 and STAT4, or IL10RA (to block autocrine IL-10), alone or in combination, and then treated with 0.1 μg/ml lipid A for 24 h. Cytokine secretion (n = 6). (B–E) MDMs were pretreated with vehicle (DMSO) or the indicated doses of either (B and C) Upadacitinib (Upa; JAK1 inhibitor) or (D and E) BMS-986165 (BMS; TYK2 inhibitor) for 1 h. (B and D) Fold induction of the indicated phosphoproteins with 0.1 μg/ml lipid A for 15 min (n = 5; similar results in an additional n = 4) (earlier time point assessed to minimize signaling through autocrine/paracrine cytokine loops). (C and E) Cells were treated with 0.1 μg/ml lipid A or 10 ng/ml IFN-γ or IL-12. Cytokines at 24 h (n = 6; similar results in an additional n = 4 for lipid A). (F) MDMs were transfected with scrambled, JAK1, TYK2, or STAT3 siRNA, and then treated with 0.1 μg/ml lipid A for 24 h. Cytokine secretion (n = 6). Mean + SEM. Significance is to scrambled siRNA–transfected, lipid A–treated cells for (F), to vehicle and cytokine- or lipid A–treated cells in (C) and (E), or as indicated. t test for (A) combined with Bonferroni–Holm correction for (B)–(F). *p < 0.05, **p < 0.01, ***p < 0.001, †p < 1 × 10−4. scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were pretreated with 5 μg/ml fludarabine (STAT1 inhibitor), 150 μM lisofylline (STAT4 inhibitor) (Cayman Chemical Company), Upadacitinib (JAK1 inhibitor), or BMS-986165 (TYK2 inhibitor) (MedChemExpress).

Techniques: Transfection, Blocking Assay