Figure 6
Figure 6. Regulation of mitochondrial biogenesis and mitochondrial targeting in CLL cells. (A) The relative gene expression of key molecules governing mitochondrial biogenesis (mitochondrial transcription factor A, tfam; nuclear respiratory factor 1, nrf1; nuclear respiratory factor 2, nrf2; estrogen-related receptor a, erra; peroxisome proliferator-activated receptor a, ppara) as analyzed in purified HD B-cells (n = 8) and CLL cells (n = 10). (B) The %al changes of the TFAM relative gene expression in purified CLL cells (n = 6) on treatment with the mitochondrial-specific antioxidant MitoQ and the general antioxidant NAC in comparison with untreated cells over 12 and 24 hours. (C) The %al changes of the HO-1 relative gene expression in purified CLL cells (n = 6) on treatment with the mitochondrial-specific antioxidant MitoQ and the general antioxidant NAC in comparison with untreated cells over 12 and 24 hours. (D) The %al changes of the TFAM relative gene expression in purified CLL cells (n = 6) on treatment with 25 μM and 50 μM of the HO-1 inhibitor tin-protoporphyrin (SnPP) over 12 and 24 hours. Bars indicate the standard error mean. (E) Effects of 17.5 µM and 35 µM PK11195 on the generation of mitochondrial ROS in CLL cells (n = 4) 1, 2, and 4 hours after its application based on the MFI of MitoSOX as quantified by FACS. (F) Respiration (OCR) is measured under basal conditions and in response to the indicated F1F0-ATPase inhibitor PK11195 in CLL cells (n = 4). The resulting effects on OCR are shown as a percentage of the baseline measurement (set as 100%). (G) A representative FACS analysis of the effects of a 24-hour PK11195 treatment on the viability of a CLL patient’s (CL71) CLL cells and T-cells and HDs’ (K5) B- and T-cells, respectively, is shown (left panel). Viable cells are 7-AAD and Annexin-V negative (highlighted box). (Right panel) the mean values of the %al specific death elicited by 17.5 µM and 35 µM PK11195 (for 24 hours) in CLL patients’ (n = 5) CLL cells and T-cells and HDs’ (n = 5) B- and T-cells. (H) Bone marrow stromal cells (eg, the HS-5 cell line) can protect CLL cells from drug-induced apoptosis. The %al specific cell death as assessed by (FACS) is shown for purified CLL cells (n = 4) treated with 35 µM PK11195 for 24 hours in the presence or absence of HS-5 cells. Bars indicate the standard error mean. Abbreviations: P, P-value; *P < .05; **P < .005; ***P < .001.

Regulation of mitochondrial biogenesis and mitochondrial targeting in CLL cells. (A) The relative gene expression of key molecules governing mitochondrial biogenesis (mitochondrial transcription factor A, tfam; nuclear respiratory factor 1, nrf1; nuclear respiratory factor 2, nrf2; estrogen-related receptor a, erra; peroxisome proliferator-activated receptor a, ppara) as analyzed in purified HD B-cells (n = 8) and CLL cells (n = 10). (B) The %al changes of the TFAM relative gene expression in purified CLL cells (n = 6) on treatment with the mitochondrial-specific antioxidant MitoQ and the general antioxidant NAC in comparison with untreated cells over 12 and 24 hours. (C) The %al changes of the HO-1 relative gene expression in purified CLL cells (n = 6) on treatment with the mitochondrial-specific antioxidant MitoQ and the general antioxidant NAC in comparison with untreated cells over 12 and 24 hours. (D) The %al changes of the TFAM relative gene expression in purified CLL cells (n = 6) on treatment with 25 μM and 50 μM of the HO-1 inhibitor tin-protoporphyrin (SnPP) over 12 and 24 hours. Bars indicate the standard error mean. (E) Effects of 17.5 µM and 35 µM PK11195 on the generation of mitochondrial ROS in CLL cells (n = 4) 1, 2, and 4 hours after its application based on the MFI of MitoSOX as quantified by FACS. (F) Respiration (OCR) is measured under basal conditions and in response to the indicated F1F0-ATPase inhibitor PK11195 in CLL cells (n = 4). The resulting effects on OCR are shown as a percentage of the baseline measurement (set as 100%). (G) A representative FACS analysis of the effects of a 24-hour PK11195 treatment on the viability of a CLL patient’s (CL71) CLL cells and T-cells and HDs’ (K5) B- and T-cells, respectively, is shown (left panel). Viable cells are 7-AAD and Annexin-V negative (highlighted box). (Right panel) the mean values of the %al specific death elicited by 17.5 µM and 35 µM PK11195 (for 24 hours) in CLL patients’ (n = 5) CLL cells and T-cells and HDs’ (n = 5) B- and T-cells. (H) Bone marrow stromal cells (eg, the HS-5 cell line) can protect CLL cells from drug-induced apoptosis. The %al specific cell death as assessed by (FACS) is shown for purified CLL cells (n = 4) treated with 35 µM PK11195 for 24 hours in the presence or absence of HS-5 cells. Bars indicate the standard error mean. Abbreviations: P, P-value; *P < .05; **P < .005; ***P < .001.

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