Figure 6.
BET inhibition synergizes with DMF in killing GCB-DLBCL cells in vivo. (A) VFN-D19 patient-derived xenograft mice were treated with either vehicle, DMF, I-BET151 or the combination of DMF and I-BET151 for 5 consecutive days (days 1-5), as indicated. Tumor volume was quantified by caliper measurements up to 10 days after start of the treatment. Each group consists of ≥7 animals. (B) Lipid peroxidation in UPF-30P cells treated with DMF, JQ1, I-BET151, or the indicated combinations was assessed by flow cytometry. The MFI of oxidized BODIPY C11 in treated cells was normalized to the MFI of the solvent control. (C) UPF-30P cells were treated with DMF alone (left panel) or in combination with I-BET151 (right panel). After 48 hours, cell numbers were determined and the combination treatment was normalized to the DMF single treatment. The CI for treatment with 10 μM DMF and 0.25 μM I-BET151 is 0.32. (D) The potential of 10 μM ferrostatin-1 (Fer-1), 100 μM α-tocopherol (α-Toco) and 10 μM Q-VD-OPh to prevent the toxicity induced by cotreatment with DMF and I-BET151 was analyzed in UPF-30P cells. (E) Transcript levels of the indicated genes in JQ1- or I-BET151-treated UPF-30P cells were quantified by qPCR. SDHA served as reference gene. (F) FSP1 protein expression in UPF-30P cells 24 hours after JQ1 or I-BET151 treatment was visualized by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase served as loading control. (G) UPF-30P cells were treated with solvent, DMF, and iFSP1 for 2 hours, as indicated. Lipid peroxidation was quantified by flow cytometry. The MFI of oxidized BODIPY C11 in treated cells was normalized to the MFI of the solvent control. (H) Survival of UPF-30P cells after 24 hours of combinatorial treatment with DMF and iFSP1 (right panel) was normalized to the DMF single treatment (left panel). The CI for treatment with 10 μM DMF and 2.5 μM iFSP1 is 0.46. (I-J) VFN-D19 patient-derived xenograft mice were treated with either vehicle, DMF, I-BET151 or the combination of DMF and I-BET151 (n = 3 for each group) for 3 consecutive days. Subsequently, the respective tumors were isolated und analyzed for AIFM2 mRNA expression by qPCR (I) or malondialdehyde levels by thiobarbituric acid reactive substances (TBARS) assay (J). Error bars correspond to the mean ± SD. Data are representative of at least 3 (B-F,H) or 2 (G) independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. CI, combination index; MFI, mean fluorescence intensity; qPCR, quantitative polymerase chain reaction.

BET inhibition synergizes with DMF in killing GCB-DLBCL cells in vivo. (A) VFN-D19 patient-derived xenograft mice were treated with either vehicle, DMF, I-BET151 or the combination of DMF and I-BET151 for 5 consecutive days (days 1-5), as indicated. Tumor volume was quantified by caliper measurements up to 10 days after start of the treatment. Each group consists of ≥7 animals. (B) Lipid peroxidation in UPF-30P cells treated with DMF, JQ1, I-BET151, or the indicated combinations was assessed by flow cytometry. The MFI of oxidized BODIPY C11 in treated cells was normalized to the MFI of the solvent control. (C) UPF-30P cells were treated with DMF alone (left panel) or in combination with I-BET151 (right panel). After 48 hours, cell numbers were determined and the combination treatment was normalized to the DMF single treatment. The CI for treatment with 10 μM DMF and 0.25 μM I-BET151 is 0.32. (D) The potential of 10 μM ferrostatin-1 (Fer-1), 100 μM α-tocopherol (α-Toco) and 10 μM Q-VD-OPh to prevent the toxicity induced by cotreatment with DMF and I-BET151 was analyzed in UPF-30P cells. (E) Transcript levels of the indicated genes in JQ1- or I-BET151-treated UPF-30P cells were quantified by qPCR. SDHA served as reference gene. (F) FSP1 protein expression in UPF-30P cells 24 hours after JQ1 or I-BET151 treatment was visualized by immunoblotting. Glyceraldehyde 3-phosphate dehydrogenase served as loading control. (G) UPF-30P cells were treated with solvent, DMF, and iFSP1 for 2 hours, as indicated. Lipid peroxidation was quantified by flow cytometry. The MFI of oxidized BODIPY C11 in treated cells was normalized to the MFI of the solvent control. (H) Survival of UPF-30P cells after 24 hours of combinatorial treatment with DMF and iFSP1 (right panel) was normalized to the DMF single treatment (left panel). The CI for treatment with 10 μM DMF and 2.5 μM iFSP1 is 0.46. (I-J) VFN-D19 patient-derived xenograft mice were treated with either vehicle, DMF, I-BET151 or the combination of DMF and I-BET151 (n = 3 for each group) for 3 consecutive days. Subsequently, the respective tumors were isolated und analyzed for AIFM2 mRNA expression by qPCR (I) or malondialdehyde levels by thiobarbituric acid reactive substances (TBARS) assay (J). Error bars correspond to the mean ± SD. Data are representative of at least 3 (B-F,H) or 2 (G) independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. CI, combination index; MFI, mean fluorescence intensity; qPCR, quantitative polymerase chain reaction.

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