|
|
Blood, Vol. 109, Issue 12, 5199-5207, June 15, 2007
Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization
Blood Muntean et al.
109: 5199
Supplemental materials for: Muntean et al, Vol 109, Issue 12, 5199-5207
Files in this Data Supplement:
- Figure S1. GATA-1 does not bind the cyclin D1 promoter in G1ER erythroid cells (PDF, 104 KB) -
Chromatin immunoprecipitations were performed using chromatin prepared from G1ER cells that express the GATA-1/ER fusion protein. Primer sets include mcyclin D1 directed against the proximal GATA site, U2C (cyclin D1 upstream control primers), NDC (cyclin D1 downstream control primers), Ubb 3′TR (ubiquitin negative control), HS2 ( -globin hypersensitive positive control) and  -globin (positive control). Each experiment was performed in the absence or presence of estrogen, which induces erythroid differentiation. Rat IgG, GATA1 (N6), and estrogen receptor (ER) antibodies were used for immunoprecipitation; none of these approaches show enrichment of GATA-1 on the cyclin D1 promoter in erythroid cells.
- Figure S2. Expression of CylinD1 and Cdk4 in retrovirally transduced cells (PDF, 79.5 KB) -
(A) Left, Western blot of protein extracted from the megakaryocytic cells lines Y10 and K562, or the Phoenix retroviral packaging cell line transfected with either empty MIGR1 or the MIGR1HA-cyclin D1 construct. Ectopic HA-cyclin D1 was detected using an anti-HA antibody. Right, Real time qPCR analysis of mRNA extracted from gradient purified wild type, GATA-1 knock down and GATA-1 knock down megakaryocytes infected with retrovirus containing empty MIGR1 or harboring the HA-cyclin D1. Expression levels are shown relative to cyclin D1 levels in wild type fetal liver cells. (B) Expression of HA-cyclin D1 and HA-Cdk4 in retroviral packaging cell line and infected primary cells. Left, western blot depicting expression of HAcyclin D1 and HA-CDK4 in the Phoenix retroviral packaging cell line following transfection with the indicated MIGR1 vectors. Megakaryocytic cell lines Y10 and K562 are shown as controls. Upper Right, real time quantitative PCR shows cyclin D1 expression in infected primary megakaryocytes, derived from fetal livers and purified by passage through a BSA gradient. mRNA was extracted from wild type, GATA-1 knock down (KD), and KD cells infected with retrovirus for the following combinations: MIGR1-E:pCS-E; CycD1:pCS-E; MIGR1-E:CDK4; and CycD1:CDK4. Expression levels are shown relative to wild type cyclin D1 levels. Lower Right, qRT-PCR analysis of Cdk4 expression in cells described above. Expression levels are shown relative to wild type Cdk4 levels.
- Figure S3. Ectopic expression of cyclin D1/Cdk 4 in wild-type megakaryocytes does not increase extent of polyploidization (PDF, 111 KB) -
Wild type megakaryocytes were differentiated in the presence of empty MigR1 and pCS (Empty) or wild-type cyclin D1 and Cdk4. Ectopic expression of cyclin D1 and Cdk4 in wild type cells did not increase polyploidization (middle panel) or cell size (right panel).
- Figure S4. Primers utilized in this study (PDF, 60.8 KB)
|
|
