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Blood, Vol. 109, Issue 2, 584-594, January 15, 2007
The THAP–zinc finger protein THAP1 regulates endothelial cell proliferation through modulation of pRB/E2F cell-cycle target genes
Blood Cayrol et al.
109: 584
Supplemental materials for: Cayrol et al, Vol 109, Issue 2, 584-594
Files in this Data Supplement:
- Figure S1. Expression of endogenous THAP1 in human tissues and ECs freshly isolated from human tonsils, rheumatoid arthritis synovium, and Crohn disease intestine (JPG, 69.5 KB)
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(A) Northern blot analysis indicates that THAP1 mRNA is not ubiquitous in human tissues. A blot of poly(A)+ RNA from multiple human tissues was hybridized with a 32P-labeled human THAP1 cDNA probe according to the manufacturer’s instructions (Clontech, Mountain View, CA). The multiple bands observed below the bona fide 2.2-kb THAP1 mRNA are specific and likely to be due to the presence of multiple polyadenylation signals (2 AATAAA and 2 ATTAAA motifs) in the THAP1 3′-untranslated region. (B) Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis shows that THAP1 mRNA is expressed in ECs freshly purified from different human tissues, including tonsils (tonsil ECs), Crohn disease intestine (Crohn ECs), and rheumatoid arthritis synovium (RA ECs), a tissue associated with high levels of angiogenesis and EC proliferation. Isolation of microvascular ECs from the different tissues and RNA analysis by RT-PCR has previously been described.1,2 Though clearly expressed by ECs in vitro and in vivo, endogenous THAP1 is not endothelial cell specific, however, since THAP1 mRNA is also detected in human Hela epithelial cells. (C) Western blot analysis reveals that endogenous THAP1 protein is expressed in proliferating ECs (HUVECs) at levels similar to those found in proliferating U2OS and Hela epithelial cancer cells in vitro. Antibodies against α-tubulin were used as control.
- Figure S2. Expression of endogenous THAP1 in relation to endothelial cell-cycle progression and cell proliferation (JPG, 127 KB)
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(A) Endogenous THAP1 mRNA is not cell-cycle regulated. HUVECs were synchronized by a thymidine block (2.5 mM, 24 hours). After release from the block, the cell-cycle profile was analyzed by flow cytometry (FACS) at the indicated time points. The histogram DNA profiles of propidium iodide–stained cells are shown at the top of the panel. Asynchronous cells are labeled AS. Quantitative PCR (qPCR) analysis was performed to determine the THAP1 mRNA relative expression level during cell-cycle progression. The transcript levels were calculated relative to GAPDH. Samples were analyzed in triplicate, and the mean and standard deviation were calculated. A single experiment is shown, representative of 2 independent experiments. (B-C) Endogenous THAP1 is cell-growth regulated. THAP1 mRNA and protein were found to be slightly up-regulated in proliferating HUVECs compared with quiescent growth-arrested HUVECs. (B) HUVECs were serum starved and restimulated to enter the cell cycle by serum addition. Determination of cell-cycle profile by flow cytometry and qPCR analysis was performed at the indicated time points after serum addition, as described in panel A. (C) Quantitative Western blot analysis was performed to compare THAP1 protein levels in quiescent (G0) and proliferating (AS) HUVECs. Relative expression levels were determined by quantitative Western blot analysis using the ECL Plex System and ImageQuant 5.0 software (GE Healthcare, Orsay, France).
REFERENCES
1. Lacorre DA, Baekkevold ES, Garrido I, et al. Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment. Blood. 2004;103:4164-4172.
2. Patterson AM, Gardner L, Shaw J, et al. Induction of a CXCL8 binding site on endothelial syndecan-3 in rheumatoid synovium. Arthritis Rheum. 2005;52:2331-2342.
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