Journal: Frontiers in immunology

Article Title: Disruption of Telomere Integrity and DNA Repair Machineries by KML001 Induces T Cell Senescence, Apoptosis, and Cellular Dysfunctions.

doi: 10.3389/fimmu.2019.01152

Figure Lengend Snippet: FIGURE 4 | KML001 impairs telomere integrity by inhibiting TRF2 protein expression. (A) Purified CD4 T cells were treated with 5 µM KML001 or DPBS control for 48 h, followed by western blotting to measure TRF2, TRF1, TIN2, POT1, TPP1, PAP1 protein expressions. Representative imaging and summary data of the western blot are shown. NS, not significant. (B) Upper panel, HCV core protein was detected by fluorescence microscopy in Huh-7.5 hepatocytes transfected with HCV-JFH1 strain, but not in the mock transfected cells. Lower panel, TRF2 was inhibited in CD4 T cells co-cultured with HCV+ Huh-7.5 cells compared to those incubated with HCV−hepatocytes. GAPDH served as a loading control. (C) Upper panel, RT-PCR detection of HIV-RNA in SupT1 cells at day 3 and day 6 of HIV-1 infection. Lower panel, western blotting of HIV-1 p24 and TRF2 expressions in SupT1 cells without (lane 1), or with low (lane 2) and high (lane 3) dose of HIV-1 infection. GAPDH served as loading control. (D,E) Western blot analysis of p53 and Sirt6 expressions in CD4 T cells exposed to KML001 (5 µM) or DPBS control for 48 h. Summary data were normalized to the DPBS control. (F) Western blot analysis of Sirt6 expression in TCR-activated CD4 T cells by anti-CD3/CD28 stimulation (1 µg/ml) and exposed to KML001 (5 µM) or DPBS treatment for 48 h. (G) Western blot analysis of p53, Sirt6, and TRF2 expressions in CD4 T cells with or without KML001 treatment in the presence or absence of proteasomal inhibitor (MG-132).

Article Snippet: Culture supernatants containing 1∼5 × 106 HIV-1 virions fromHEK293T (human embryonic kidney 293 cells transformed with SV40 large T antigen, purchased from the American Type Culture Collection) cells transfected with pNL4-3 plasmid were employed to infect SupT1 (a human CD4T cell line derived from NIH AIDS Reagent Program) using the spinoculation method (37).

Techniques: Expressing, Control, Western Blot, Imaging, Microscopy, Transfection, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: Frontiers in immunology

Article Title: Disruption of Telomere Integrity and DNA Repair Machineries by KML001 Induces T Cell Senescence, Apoptosis, and Cellular Dysfunctions.

doi: 10.3389/fimmu.2019.01152

Figure Lengend Snippet: FIGURE 6 | CD4 T cells derived from HCV or HIV-infected individuals are more vulnerable to telomere loss-mediated apoptosis. (A,B) Telomere length, measured was by Flow-FISH, in CD4 T cells isolated from HCV- or HIV-infected individuals and HS. Representative overlaid histograms and summary data with mean ± SEM and p-values are shown. (C) Representative overlaid histogram and summary data of Flow-FISH analysis for telomere length in SupT1 cells infected with or without HIV-1 for 3 and 6 days. The mean fluorescence intensity (MFI) of telomere length in HIV-infected cells are normalized by those in uninfected cells. (D) PBMCs derived from chronically HCV-infected individuals were treated with 5 µM KML001 or DPBS control for 24 h, followed by flow cytometry analysis of Av expression in CD4+ T cells. (E) PBMCs derived from latently HIV-infected individuals and HS were treated with 5 µM KML001 or DPBS control for 24 h, followed by flow cytometry analysis of Av expression in CD4+ T cells. Summary data with mean ± SEM (n = 8) and p-values are shown. (F) Dose-dependent induction of apoptotic death of CD4 T cells isolated from HCV- and HIV-infected individual and HS (n = 3 in each group) and exposed to varying concentrations of KML001 for 12 h. Representative dot plots of CD4+ T cells at 2 µM KML001 treatment and summary data of the dynamic changes of Av/7AAD levels in CD4+ T cells following varying concentrations of KML001 treatment are shown.

Article Snippet: Culture supernatants containing 1∼5 × 106 HIV-1 virions fromHEK293T (human embryonic kidney 293 cells transformed with SV40 large T antigen, purchased from the American Type Culture Collection) cells transfected with pNL4-3 plasmid were employed to infect SupT1 (a human CD4T cell line derived from NIH AIDS Reagent Program) using the spinoculation method (37).

Techniques: Derivative Assay, Infection, Isolation, Control, Cytometry, Expressing