Fig 2: Top1mt expression and activity are inhibited in CD4 T cells during chronic viral infection. (A) Top1mt protein (green), MitoTracker (mt, red), and their colocalization (yellow) in mitochondria of CD4 T cells from HCV- and HIV-infected patients and healthy subjects (HS). The nucleus was stained with DAPI, and representative confocal microscopy images were merged. (B) Top1mt protein expression in CD4 T cells from HCV- and HIV-infected patients and HS. Representative images and summary data of Western blots are shown (n=10). The Top1mt band intensity was normalized by ß-Actin. (C) Top1 activity in mitochondria of CD4 T cells from HCV- and HIV-infected patients versus HS. Representative images and summary data of Top1-mediated digestion of supercoiled DNA substrate are shown. (D) Top1cc was detected in mtDNA from CD4 T cells from HCV- and HIV-infected patients versus HS. Representative images and summary data of Top1cc band intensity (normalized to HS) are shown.

Fig 3: Top1 inhibition by CPT induces topological mtDNA damage in healthy CD4 T cells. (A) Top1mt protein (green), MitoTracker (mt, red), and their colocalization (yellow) in mitochondria in HS-CD4 T cells treated with DMSO or CPT (10 µM) for 24 h and observed by confocal microscopy. The nuclei were stained with DAPI, and representative confocal microscopy images were merged. (B) Immunoblotting of Top1mt in HS-CD4 T cells treated with CPT (5 µM or 10µM) or DMSO for 24 h, 48 h or 72 h. (C) Immunoblotting and summary data of Top1mt in HS-CD4 T cells treated with 10µM CPT or DMSO for 24 h. Representative images and summary data from independent experiments (n=4) are shown. (D) Top1 activity and summary data in mitochondria and cytosol in HS-CD4 T cells treated with CPT (5 µM) or DMSO for 24 h, determined by Top1 activity assay (n=7). (E) Immunoblotting and summary data of Top1cc in mtDNA from HS-CD4 T cells exposed to CPT (5 µM) or DMSO for 48 h (n=8). (F) Immunoblotting and summary data of PARP1 in mitochondria from HS-CD4 T cells treated to CPT (5 µM) or DMSO for 48 h (n=7). (G) mtDNA copy numbers relative to nuclear DNA were determined by real-time RT-PCR in HS-CD4 treated with CPT (5 µM) or DMSO for 24 h (n=8).

Fig 4: Top1 inhibition by CPT induces programmed cell death in healthy CD4 T cells via multiple cell death pathways. Healthy CD4 T cells were treated with CPT (5 µM) or DMSO for 24 h, followed by flow cytometry analysis of the expression levels of Caspase-3 (A), Caspase-1 (B), GPX4 (C), pBAD (D), Mcl1 (E), Survivin (F), RIPK1 (G), and RIPK3 (H).

Fig 5: Top1 inhibition by CPT or cellular oxidative stress induces mitochondrial dysfunction in healthy CD4 T cells. (A) Oxygen consumption rate (OCR) for non-mitochondrial oxygen consumption, basal respiration, maximal respiration, proton leak, ATP production, and spare respiratory capacity in HS-CD4 T cells treated with CPT (5 µM) or DMSO for 48 h. Representative images and summary data from independent experiments are shown (n=7). (B) Western blot analysis of Top1mt levels (normalized to ß-Actin) in E6-1 cells transfected with/mito-FAP-mCer3 cells and treated with or without MG2I dye plus light for 10-20 min (n=5). (C) Top1 enzymatic activity in mitochondria from E6-1/mito-FAP-mCer3 cells treated with or without MG2I dye plus light for 10 or 20 min (n=8). (D) Representative Western blots and summary data of IRF3, and representative data of total PARP1/cleaved PARP1 levels in mitochondria from E6-1/mito-FAP-mCer3 transfected cells treated with or without MG2I dye plus light for 10 or 20 min (n=6). (E) Summarized western blotting data of total PARP1, and cleaved PARP1 levels in mitochondria from E6-1/mito-FAP-mCer3 transfected cells treated with or without MG2I dye plus light for 10 or 20 min (n = 4) *p < 0.05, **p < 0.01, ***p < 0.001.