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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Mol Cancer Ther. 2016 Mar 4;15(6):1353–1363. doi: 10.1158/1535-7163.MCT-15-0332

Figure 2. ATF3 knockdown via siATF3 inhibits apoptosis in prostate cancer cells after AD and edelfosine treatment.

Figure 2

LNCaP cells were grown for 3 days in complete or AD medium and treated with edelfosine (5 μM and 10 μM) alone or in combination with AD for Annexin V staining. (A) Treated cells were harvested after 24 h and stained using the Guava Nexin kit. Percentage of Annexin V positive cells was measured by flow cytometric analysis and the data processed using CytoSoft software. (B) LNCaP and (C) VCaP cells were harvested 24 hours following treatment and caspase 3/7 activity was determined using the Apo-ONE™ Homogeneous caspase 3/7 assay kit. For ATF3 knockdown, both LNCaP and VCaP cells grown in complete or AD medium were transfected with siATF3 (10 nM), followed by treatment with edelfosine (5 or 10 μM). (D) LNCaP and VCaP cells were grown as described above and were treated with AD and/or edelfosine (0, 2.5, 5 and 10 μM). Treated cells were harvested after 24 h; total proteins were examined by Western immunoblotting for ATF3 expression levels. The same blot was stripped and re-probed for β-actin. (E) ATF3 knockdown via siATF3 (10 nM), after edelfosine (10 μM) treatment was confirmed by Western blot analysis using AFT3 specific antibodies. (F) LNCaP cells were transfected with control siRNA or siATF3 (10 nM), followed by edelfosine (10 μM) treatment, caspase 3/7 activity was measured. Data shown represent the mean ± SD from three independent experiments. *p < 0.05, compared to respective control, **p < 0.05, compared to edelfosine 5 μM (one way ANOVA, Bonferroni test).