Figure 5.
Figure 5. Pharmacologic inhibition of JNK and transfection of JNK1 siRNA significantly protect cells from apoptosis induced by coadministration of 2-ME and the HDACIs, NaB and SAHA. U937 cells were pretreated with 10 μM JNK inhibitor, SP600125 (SP), for 1 hour, followed by the addition of 0.5 μM 2-ME combined with either NaB (1 mM) or SAHA (1.5 μM) for 24 hours. (A) Cells were stained with annexin V/PI, and apoptosis was determined using flow cytometry as described in “Materials and methods.” The values obtained from annexin V/PI assays represent the mean ± SD for 3 separate experiments. *Values for cells cotreated with 2-ME, NaB or SAHA, and SP are significantly less than those obtained for cells treated with 2-ME and NaB or SAHA (Student t test; P < .01). (B) After treatment, total cellular extracts were prepared and subjected to Western blot analysis using antibodies against phospho-c-Jun, c-Jun. (C) After treatment, total cellular or cytosolic extracts were prepared and subjected to Western blot analysis using antibodies against PARP, full-length caspase-8 and caspase-9, cleaved caspase-3, AIF, and cytochrome c, respectively. U937 cells were transfected with JNK1 siRNA oligonucleotides or controls and incubated for 24 hours at 37°C, after which cells were treated with 0.5 μM 2-ME in the presence of 1 mM of NaB or 1.5 μM of SAHA for 24 hours. (D) After treatment, total cellular extracts were prepared and subjected to Western blot analysis using antibodies against phospho-JNK, JNK1, and JNK2. (E) Apoptosis was determined using the annexin V-FITC/PI assay as described in “Materials and methods.” *Values for cells treated with 2-ME in the presence of NaB or SAHA after transfection with JNK1 siRNA oligonucleotides are significantly decreased compared with those for control cells treated with 2-ME in the presence of NaB or SAHA (Student t test; P < .01). For Western blot assay, each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading. Two additional studies yielded equivalent results.

Pharmacologic inhibition of JNK and transfection of JNK1 siRNA significantly protect cells from apoptosis induced by coadministration of 2-ME and the HDACIs, NaB and SAHA. U937 cells were pretreated with 10 μM JNK inhibitor, SP600125 (SP), for 1 hour, followed by the addition of 0.5 μM 2-ME combined with either NaB (1 mM) or SAHA (1.5 μM) for 24 hours. (A) Cells were stained with annexin V/PI, and apoptosis was determined using flow cytometry as described in “Materials and methods.” The values obtained from annexin V/PI assays represent the mean ± SD for 3 separate experiments. *Values for cells cotreated with 2-ME, NaB or SAHA, and SP are significantly less than those obtained for cells treated with 2-ME and NaB or SAHA (Student t test; P < .01). (B) After treatment, total cellular extracts were prepared and subjected to Western blot analysis using antibodies against phospho-c-Jun, c-Jun. (C) After treatment, total cellular or cytosolic extracts were prepared and subjected to Western blot analysis using antibodies against PARP, full-length caspase-8 and caspase-9, cleaved caspase-3, AIF, and cytochrome c, respectively. U937 cells were transfected with JNK1 siRNA oligonucleotides or controls and incubated for 24 hours at 37°C, after which cells were treated with 0.5 μM 2-ME in the presence of 1 mM of NaB or 1.5 μM of SAHA for 24 hours. (D) After treatment, total cellular extracts were prepared and subjected to Western blot analysis using antibodies against phospho-JNK, JNK1, and JNK2. (E) Apoptosis was determined using the annexin V-FITC/PI assay as described in “Materials and methods.” *Values for cells treated with 2-ME in the presence of NaB or SAHA after transfection with JNK1 siRNA oligonucleotides are significantly decreased compared with those for control cells treated with 2-ME in the presence of NaB or SAHA (Student t test; P < .01). For Western blot assay, each lane was loaded with 30 μg protein; blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading. Two additional studies yielded equivalent results.

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