American Society of Hematology

Figure 5

$Figure 5. AIF-induced nuclear condensation and large-scale DNA fragmentation of DLBCL cells exposed to E7123. (A) Immunoblot analyses of AIF in cytosolic fraction extracts of HT, DB, and KARPAS-422 cells exposed to 60μM of E7123 during 4 hours with or without pretreatment with 1 hour 50μM ZVAD or 30 minutes 10μM CsA. The remaining extract containing mitochondrial and nuclear fractions of vehicle-treated cells was used to control (C) for the presence of the evaluated proteins in the mitochondria. The absence of COX-II expression indicated no mitochondrial contamination of the cytoplasmic fraction. GAPDH was used as a cytoplasmic fraction marker, as well as a control for protein loading. (B) Hoechst staining of DB and KARPAS-422 cells after 4-hour exposure to 60μM E7123 with or without pretreatment with ZVAD 50μM for 1 hour or CsA 10μM during 30 minutes (original magnification ×400). Slides were viewed with an inverted microscope (Axiovert 200M). Images were acquired using a digital camera (Coolsnap; Photometrics) and processed with MetaMorph Version 5.01 software. (C) Pulse-field gel electrophoresis of DB, HT, and KARPAS-422 cells treated with vehicle or 60μM E7123 during 4 hours. Pretreatment with 10μM of CsA during 30 minutes blocked the 50-kb pair DNA fragments induced by E7123.$

AIF-induced nuclear condensation and large-scale DNA fragmentation of DLBCL cells exposed to E7123. (A) Immunoblot analyses of AIF in cytosolic fraction extracts of HT, DB, and KARPAS-422 cells exposed to 60μM of E7123 during 4 hours with or without pretreatment with 1 hour 50μM ZVAD or 30 minutes 10μM CsA. The remaining extract containing mitochondrial and nuclear fractions of vehicle-treated cells was used to control (C) for the presence of the evaluated proteins in the mitochondria. The absence of COX-II expression indicated no mitochondrial contamination of the cytoplasmic fraction. GAPDH was used as a cytoplasmic fraction marker, as well as a control for protein loading. (B) Hoechst staining of DB and KARPAS-422 cells after 4-hour exposure to 60μM E7123 with or without pretreatment with ZVAD 50μM for 1 hour or CsA 10μM during 30 minutes (original magnification ×400). Slides were viewed with an inverted microscope (Axiovert 200M). Images were acquired using a digital camera (Coolsnap; Photometrics) and processed with MetaMorph Version 5.01 software. (C) Pulse-field gel electrophoresis of DB, HT, and KARPAS-422 cells treated with vehicle or 60μM E7123 during 4 hours. Pretreatment with 10μM of CsA during 30 minutes blocked the 50-kb pair DNA fragments induced by E7123.

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