Figure 1

Vorinostat does not require a functional autophagic pathway to induce tumor cell death in Eμ-myc/Apaf-1^−/− lymphomas. (a) Eμ-myc/Apaf-1^−/− lymphomas were retrovirally transduced with constructs expressing shRNAs targeting Atg5 (clone 1472 and 1389, respectively), Atg7 (clone 698) or a scrambled control. Knockdown efficiency was tested via western blot using antibodies specific for Atg5 and Atg7 (Cell Signaling Technologies, Inc., Danvers, MA, USA, no. 2630 and 2631). Anti-β-actin was used as a loading control. Results are a representative of at least three separate westerns. Atg5-shRNA-expressing Eμ-myc/Apaf-1^−/− (1389 and 1472), Atg7-shRNA-expressing Eμ-myc/Apaf-1^−/− (698) and control-shRNA-expressing Eμ-myc/Apaf-1^−/− lymphomas were treated with 1 μM vorinostat (V) or DMSO (D) for 24 h and LC3 processing was determined by western blot using antibody specific for LC3 (NanoTools Antikoerpertechnik GmbH & Co. KG., Teningen, Germany, no. 0260-100/LC3-2G6). Anti-p44/42 ERK (Cell Signaling Technology, Inc., Danvers, MA, USA, no. 9107) or anti-β-actin were used as loading controls. Quantitative western analysis was performed using ImageJ software (public domain software by Wayne Rasband), National Institute of Health, USA. Results are representative of at least three separate westerns. (b) Eμ-myc, Eμ-myc/Apaf-1^−/− and Eμ-myc/Apaf-1^−/− lymphomas expressing the various shRNA constructs were treated with increasing concentrations of vorinostat for 24 and 48 h. Cell viability was assessed by annexin V/propidium iodide staining and error bars indicate ±S.E.M. of at least three independent experiments. (c) C57Bl/6 mice bearing Atg5-shRNA-expressing Eμ-myc/Apaf-1^−/− (1389), Atg7-shRNA-expressing Eμ-myc/Apaf-1^−/− (698) or control-shRNA-expressing Eμ-myc/Apaf-1^−/− lymphomas were treated with one dose of vorinostat (200 mg/kg). Spleen and lymph nodes were harvested at the indicated time points (h) following HDACi treatment and the percentage of lymphoma cells in lymph nodes was determined using flow cytometry in the presence of propidium iodide. Each time point is representative of 2–3 mice