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: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Antiviral activity of apoptosis-inducing Bcl-2 inhibitors. (A) Virus propagation in the presence of OLX or ABT-737. A549 cells were treated with serial dilutions of each compound starting 2 h prior to infection (MOI = 0.01) with rLCMV/ZsG. At 48 hpi, ZsG expression levels were measured and normalized to vehicle control (dimethyl sulfoxide [DMSO])–treated controls. (B) Cell viability was determined using the CellTiter-Glo assay after 48 h of compound treatment. The results were normalized to vehicle (DMSO)–treated controls. (C) EC 50 , CC 50 , and selectivity index (SI; CC 50 /EC 50 ) were calculated for OLX and ABT-737.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: Activity Assay, Infection, Expressing, Glo Assay

Journal: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Effects of Bcl-2 inhibitor-induced apoptosis on LCMV multiplication. (A) A549 cells were treated with the indicated compounds and concentrations for 48 h. Harvested cells were incubated with annexin V conjugate (Alexa Fluor 488) and 7-aminoactinomycin D (7-AAD) and analyzed by flow cytometry. The plots are the representative gatings of each compound (left panels). The graph reveals annexin V–positive cell populations (Q2 + Q3) (right panel). (B) Whole-cell lysates were collected at the indicated times, and caspase-3/7 activity was determined and normalized to total protein amount in the cell lysate. (C) A549 cells were treated with the indicated compounds 2 h before infection with rLCMV/ZsG. At 48 hpi, ZsG expression levels were determined and normalized to vehicle-treated control. (D) A549 cells were treated with the compounds for 2 h followed by infection with rLCMV/ZsG. At 48 hpi, caspase-3/7 activity was determined in whole-cell lysates, and values were normalized to total protein in cell lysates. Treatment concentrations were as follows: obatoclax (0.1 µM), ABT-737 (2 µM), staurosporine (1 µM), ribavirin (100 µM), z-VAD-FMK (100 µM), and Z-DEVD-FMK (100 µM). Cmp, compound; RLU, relative light units.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: Incubation, Flow Cytometry, Activity Assay, Infection, Expressing

Journal: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Effects of Bcl-2 inhibitor-induced G0/G1 arrest on LCMV multiplication. (A) A549 cells were treated with the indicated compounds and concentrations for the indicated times, followed by addition of BrdU to the cell culture medium for 1 h before harvesting cells. Cell cycle positions and active DNA synthesis in fixed cells were analyzed by the correlated levels of total DNA and incorporated BrdU using flow cytometry. (i) Plots are representative gatings for each compound treatment. (ii) Cells (%) at each phase of the cell cycle after 24 and 48 h of treatment with each compound. (B) Whole-cell lysates were collected at 48 h posttreatment, and protein expression levels were determined by Western blotting. (C, D) 293T cells were transfected with the indicated siRNAs and for 4 h later infected (MOI = 0.01) with rLCMV/ZsG. At 48 hpi, levels of infectious progeny in tissue culture supernatants (C) and protein expression levels (D) were determined by FFA and Western blotting, respectively. N.C, negative control. (E) A549 cells were pretreated with the indicated compounds and infected with rLCMV/ZsG. At 48 hpi, the cell cycle was analyzed using the BrdU assay. Treatment was as follows: OLX (0.1 µM), ABT-737 (2 µM), dinaciclib (5 µM), and ribavirin (100 µM). Statistical significance was calculated by analysis of variance (ANOVA). *, P < 0.05; **, P < 0.002; ***, P < 0.0002; ****, P < 0.00001. LCMV, lymphocytic choriomeningitis virus.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: Cell Culture, DNA Synthesis, Flow Cytometry, Expressing, Western Blot, Transfection, Infection, Negative Control, BrdU Staining

Journal: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Effects of Bcl-2 inhibitors on LCMV RNA synthesis at early times of infection. (A, B) After 24 h of pretreatment of the compounds, A549 cells were infected with rLCMV/ZsG (MOI = 3), and then the compounds were readded. At the indicated time points, total cellular RNA was purified. Equal amounts of RNA (1 µg) were used to generate cDNA using strand-specific primers. Genomic (A) and antigenomic strands (B) of cDNA were used in qPCR to determine the levels of viral nucleoprotein (NP) gene. Treatment was as follows: OLX (0.1 µM), ABT-737 (2 µM), and ribavirin (100 µM). Statistical significance was calculated by ANOVA. *, P < 0.05; **, P < 0.002; ***, P < 0.0002; ****, P < 0.00001.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: Infection, Purification

Journal: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Antiviral effects of the Bcl-2 inhibitor in vivo . (A) Adult C57BL/6 mice ( n = 4/group) were treated with ABT-737 (indicated doses; intraperitoneal route [IP]) or vehicle for 7 days, and body weight changes were monitored daily. (B, C) Adult C57BL/6 mice ( n = 4/group) were treated with ABT-737 (20 mg/kg/day; IP) or vehicle for 17 days. At day 0, the mice were infected with rCl-13 (2 × 10 6 PFU/mouse). Body weight changes (B) and serum virus titers (C) were determined at the indicated time points. Statistical significance was calculated by ANOVA. *, P < 0.05; **, P < 0.002; ***, P < 0.0002; ****, P < 0.00001. FFU, focus-forming units; P.I., postinfection; VC, vehicle control.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: In Vivo, Infection

Journal: Journal of Virology

Article Title: Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase

doi: 10.1128/JVI.01399-21

Figure Lengend Snippet: Broad-spectrum antiviral activity of Bcl-2 inhibitors. (A to C) Normalized virus propagation in the presence of OLX or ABT-737. Vero cells were treated with serial dilutions of each compound starting 2 h prior to infection with r3Can/GFP (A), r3ML29/GFP (B), or SARS-CoV-2 (C). At 72 hpi, GFP expression levels in infected cells (A, B) or titers of infectious SARS-CoV-2 in tissue culture supernatants as determined by IFFA (C) were measured and normalized to vehicle (DMSO)–treated controls.

Article Snippet: For the in vivo test, ABT-737 was purchased from AstaTech Inc. (Bristol, PA).

Techniques: Activity Assay, Infection, Expressing