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Blood, Vol. 113, Issue 17, 4038-4048, April 23, 2009
Cotreatment with BCL-2 antagonist sensitizes cutaneous T-cell lymphoma to lethal action of HDAC7-Nur77–based mechanism
Blood Chen et al.
113: 4038
Supplemental materials for: Chen et al
Files in this Data Supplement:
- Table S1. Primer sequences used in this study (PDF, 47.4 KB)
- Figure S1. Treatment with panobinostat alters the expression levels of apoptosis regulators in panobinostat sensitive HH and HuT78 but not panobinostat insensitive MJ CTCL cells (JPG, 42.3 KB)
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(A) HuT78 cells were treated with the indicated concentrations of panobinostat for 24 hours. Total cell lysates were harvested, and immunoblot analysis was performed to assess cleavage of Caspase 3 and PARP. The expression of β-actin in the lysates served as the loading control. (B) HH and MJ cells were treated with 20 nmol/L of panobinostat for the indicated time intervals. Then total cell lysates were prepared, and immunoblot analysis was performed for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak, Bim, and Bid. The expression levels of α-tubulin in the lysates served as the loading control. (C) HH and HuT78 cells were treated with 20 nmol/L of panobinostat for the indicated time intervals. Following this, total cell lysates were harvested, and immunoblot analysis was performed for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak, Bim, and Bid. The expression levels of α-tubulin in the lysates served as the loading control.
- Figure S2. Panobinostat depletes HDAC7, induces Nur77 and Nor1 in a time dependent manner in sensitive but not resistant CTCL cells (JPG, 64.7 KB)
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(A) HH and MJ cells were treated with 20 nmol/L of panobinostat for the indicated times then total RNA were isolated, and RT-PCR was done for HDAC7 Nur77 and Nor1. A β-actin–specific reaction and expression levels served to control for equal loading. Alternatively, cell lysates were prepared, and immunoblot analysis was done for HDAC7, Nur77, and acetylated α-tubulin. The expression levels of β-actin in the lysates served as the loading control. (B) HuT78 cells were treated with the indicated concentrations of panobinostat of 4 hours. Following this, total RNA was isolated and RT-PCR was done for HDAC7, Nur77, and Nor1. A β-action–specific reaction and expression levels served to control for equal loading.
- Figure S3. Downregulation of HDAC7 by siRNA increases cell death in HH cells (JPG, 22.5 KB)
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(A) HH cells were transfected with control or HDAC7 specific siRNA for 48 hours. Following this, cells were harvested and total RNA was extracted and RT-PCR was done for HDAC7. A β-action–specific PCR reaction and expression levels served as a control for equal loading. Alternatively, total cell lysates were prepared and immunoblotted for HDAC7. The expression levels of β-actin in the lysates served as the loading control. (B) HH cells were transfected with the control or HDAC7 siRNA. Following an interval of 96 hours, loss of cell viability was assessed by trypan blue dye uptake method utilizing a hemocytometer.
- Figure S4. Panobinostat treatment or overexpression of Nur77 does not induce mRNA levels of TRAIL and FasL in CTCL cells (JPG, 32.8 KB)
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(A) HH cells were treated with the indicated concentrations of panobinostat for 4 hours. Then total RNA was isolated, and RT-PCR was done for TRAIL and FasL. A β-action–specific reaction and expression levels served to control for equal loading. (B) HH cells were transfected with empty vector or pcDNA3.1 FLAG/Nur77 for 48 hours. Following this, total RNA was isolated, and qRT-PCR was done for TRAIL and FasL. A β-action–specific reaction and expression served to control for equal loading.
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