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Blood, Vol. 114, Issue 2, 380-393, July 9, 2009
The histone deacetylase inhibitors LAQ824 and LBH589 do not require death receptor signaling or a functional apoptosome to mediate tumor cell death or therapeutic efficacy
Blood Ellis et al.
114: 380
Supplemental materials for: Ellis et al
Preparation of whole cell extracts. Cell pellets were resuspended in lysis buffer (0.15 M NaCl, 10 mM Tris-Cl pH 7.4, 5 mM EDTA, 1% Triton X-100) supplemented with protease inhibitors (Complete Protease Inhibitor Cocktail, Roche) and incubated on ice for 30 min with repeated vortexing. Lysates were cleared by centrifugation (13000 rpm, 15 min, 4°C), protein levels were quantified using a Bradford protein quantification assay (Pierce) and 30 µg lysate was used for Western Blotting analysis. Western blotting. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and electroblotted on PVDF membranes (Millipore). For analysis of histone acetylation, membranes were incubated with anti-CrmA, anti–Bcl-2, anti–Bcl-XL, anti-LC3 (2G6, Nanotools), anti-p21 (F5, Santa Cruz), or anti-p42 (3A7, Cell Signaling Technology) antibody (1:1000 in PBS/0.05% Tween-20) followed by subsequent incubation with horseradish peroxidase-conjugated secondary antibodies (DAKO). Immunoreactive bands were visualized by enhanced chemiluminescence (Amersham). Intracellular cytochrome c detection. Cells were resuspended in ice-cold PBS containing 120mM KCl and 50ug/ml digitonin and left on ice for 2 min. Permeabilized cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed in PBS and incubated for 1 hour in blocking buffer (3% BSA, 0.05% saponin in PBS). Cells were then incubated overnight with anti-cytochrome c (6H2.B4, BD Biosciences) antibody at 4°C, washed in blocking buffer and incubated at room temperature for 1 hour with PE-conjugated anti-mouse IgG. Cells were then analysed by flow cytometry for detection of cytochrome c in the mitochondria.
Files in this Data Supplement:
- Figure S1. C57BL/6 mice bearing an independently-derived Eµ-myc lymphoma (Eµ-myc#2) different to that used in Fig. 1 of the main text were treated i.v. with LAQ824 (75mg/kg) or LBH589 (80mg/kg) and lymph nodes were harvested at the indicated times (JPG, 215 KB)
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Sections were assessed by H and E staining (top panel), immunohistochemistry using an anti-acetyl histone H3 antibody (middle panel) and TUNEL staining (bottom panel). Each time point is an individual mouse.
- Figure S2 (JPG, 130 KB)
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(A) Whole cell lysates were prepared from Eµ-myc, Eµ-myc/CrmA, and Eµ-myc/CrmA#2 (an independently-derived Eµ-myc/CrmA lymphoma different to that used in Fig. 2 of the main text) lymphomas. Western blot analysis was performed with anti-CrmA antibody. Equivalent protein loading was confirmed by probing for β-actin. (B) C57BL/6 mice bearing palpable Eµ-myc/CrmA#2 lymphomas were treated i.v. with LAQ824 (75mg/kg) or LBH589 (80mg/kg) and lymph nodes were harvested at the indicated times. Sections were assessed by H and E staining (top panel), immunohistochemistry using an anti-acetyl histone H3 antibody (middle panel) and TUNEL staining (bottom panel). Each time point is an individual mouse.
- Figure S3 (JPG, 147 KB)
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(A) Whole cell lysates were prepared from Eµ-myc/bcl-2 and Eµ-myc/bcl-XL lymphomas used in Fig. 3 of the main text. Western blot analysis was performed with anti–Bcl-2 or anti–Bcl-XL antibodies. Equivalent protein loading was confirmed by probing for β-actin. An independently-derived Eµ-myc/bcl-2 lymphoma (Eµ-myc/bcl-2#2) was assessed by intracellular FACs using and antibody against Bcl-2 (red line) an isotype control antibody (blue line) or no primary antibody. Primary antibody binding was assessed using APC-conjugated anti-mouse secondary antibody. (B) C57BL/6 mice bearing palpable Eµ-myc/bcl-2#2 lymphomas were treated i.v. with LAQ824 (75mg/kg) or LBH589 (80mg/kg) and lymph nodes were harvested at the indicated times. Sections were assessed by H and E staining (top panel), immunohistochemistry using an anti-acetyl histone H3 antibody (middle panel) and TUNEL staining (bottom panel). Each time point is an individual mouse.
- Figure S4 (JPG, 89.8 KB)
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(A) Eµ-myc, Eµ-myc/bcl-2 and Eµ- myc/bcl-XL lymphomas were left untreated (U) or treated with (i) 4 or 10nM LBH589 (L4 or L10) or vehicle (V) or (ii) 25 or 50nM LAQ824 (L25 or L25) or vehicle (V) for 6hr and analysed for endogenous p21 by western blot (upper panels). Arrows indicate positions of p21WAF1/CIP1. Membranes were stripped and re-probed for p42 as loading control (bottom panels). (B) Eµ-myc and Eµ-myc/Apaf-1−∕− lymphomas were left untreated (U) or treated with 4 or 10nM LBH589 (L4 or L10) or 25 or 50nM LAQ824 (L25 or L25) or vehicle (V) for 6hr and analysed for endogenous p21WAF1/CIP1 by western blot (upper panels). Arrows indicate positions of p21. Membranes were stripped and re-probed for p42 as loading control (bottom panels).
- Figure S5 (JPG, 195 KB)
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C57BL/6 mice bearing palpable Eµ-myc/p53−∕−#2 lymphomas (an independently-derived Eµ-myc/p53−∕− lymphoma different to that used in Fig. 4 of the main text) were treated i.v. with LAQ824 (75mg/kg) or LBH589 (80mg/kg) and lymph nodes were harvested at the indicated times. Sections were assessed by H and E staining (top panel), immunohistochemistry using an anti-acetyl histone H3 antibody (middle panel) and TUNEL staining (bottom panel). Each time point is an individual mouse.
- Figure S6 (JPG, 211 KB)
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(A) Cells were left untreated or treated with LBH589 (4 and 10nM) or vehicle for 8hr. (i) Cells were permeabilised and stained for mitochondrial cytochrome C by intracellular FACS staining. Cells stained for cytochrome C are considered to have intact mitochondria. (ii) the bar diagrams summarises three independent experiments and shows mean +∕− SE. (B) Cells were left untreated or treated with LAQ824 (25 and 50nM) or vehicle for 8hr. (i) Cells were permeabilised and stained for mitochondrial cytochrome C by intracellular FACS staining. Cells stained for cytochrome C are considered to have intact mitochondria. (ii) the bar diagrams summarises three independent experiments and shows mean +∕− SE.
- Figure S7 (JPG, 175 KB)
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C57BL/6 mice bearing palpable Eµ-myc/apaf-1−∕−#2 lymphomas (an independently-derived Eµ-myc/apaf-1−∕− lymphoma different to that used in Fig. 4 of the main text) were treated i.v. with LAQ824 (75mg/kg) or LBH589 (80mg/kg) and lymph nodes were harvested at the indicated times. Sections were assessed by H and E staining (top panel), immunohistochemistry using an anti-acetyl histone H3 antibody (middle panel) and TUNEL staining (bottom panel). Each time point is an individual mouse.
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