Expression of Prostacyclin Receptor in Human Megakaryocytes by Yutaka Sasaki, Takayuki Takahashi, Issei Tanaka, Kishiko Nakamura, Yoshiaki Okuno, Osamu Nakagawa, Shuh Narumiya, and Kazuwa Nakao Blood Volume 90(3):1039-1046 August 1, 1997 ©1997 by American Society of Hematology

Northern blot analysis of PGI2-R mRNA in human hematopoietic cell lines. Northern blot analysis of PGI2-R mRNA in human hematopoietic cell lines. A total of 20 μg of total RNA from respective cell lines was electrophoresed in 1.5 % agarose-formaldehyde gel and hybridized with 1.9 kb EcoRI fragment of phIPR1 for PGI2-R cDNA. The 18S ribosomal RNA was used as the internal control. Lane 1, MOLT4; lane 2, MM-S1; lane 3, U266; lane 4, THP-1; lane 5, HL60; lane 6, KU812; lane 7, HEL; lane 8, NS-Meg; lane 9, CMK11-5; lane 10, CMK; lane 11, Meg-01; lane 12, K562; lane 13, JK-1. The experiment is representative of three performed. Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Effect of PMA on the expression of PGI2-R, PF4, and GPIIb mRNA in HEL, NS-Meg, and CMK11-5 cells. Effect of PMA on the expression of PGI2-R, PF4, and GPIIb mRNA in HEL, NS-Meg, and CMK11-5 cells. PMA was added at the initiation of culture and cells harvested at the indicated times were examined by Northern analysis. The figure shows the representative results from triplicate experiments repeated twice. PMA enhanced PGI2-R expression in all of these cell lines. The time and intensity of maximum PGI2-R expression was as follows: HEL, 48 hours (threefold), NS-Meg, 48 hours (fivefold), and CMK11-5, 72 hours (eightfold). PMA enhanced the expression of GPIIb mRNA in these cell lines and that of PF4 mRNA in HEL cells. The controls were the cells cultured without PMA. They yielded no enhancement of PGI2-R for the entire culture periods except a little enhancement in HEL at 48 hours (48-hour controls are shown). All of the PMA-stimulated expressions were significantly different from respective control expressions in all of these cell lines (P < .01). Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Analysis of the Northern blottings in Fig 3A and B by densitometry. Analysis of the Northern blottings in Fig 3A and B by densitometry. Each value (mean ± SE, n = 3) shows the ratio of PGI2-R expression after 24 or 48 hours of culture versus the respective control expression. *1, P < .05; *2, P < .01; *3, P < .001 as compared with the respective controls. Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Effect of PMA and TPO on PGI2-R expression. Effect of PMA and TPO on PGI2-R expression. HEL () and NS-Meg (▪) cells were preincubated for 48 hours with or without (control) either PMA or TPO. PGI2-R expression was evaluated by specific [3H]iloprost binding to the cells. Values (mean ± SE, n = 4) are expressed as a ratio of scintillation count of the control cells (nontreated HEL, 1.3 × 105 ± 5.3 × 103 dpm/1 × 106 cells, nontreated NS-Meg, 4.2 × 104 ± 3.9 × 103). Preincubation with PMA or TPO increased the binding capacity of the ligand up to sixfold and threefold in HEL cells and fivefold and twofold in NS-Meg cells, respectively, when compared with control values. The experiment is representative of three performed. Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology

Expression of PGI2-R and PF4 mRNA in normal human megakaryocytes as evaluated by semiquantitative RT-PCR. Expression of PGI2-R and PF4 mRNA in normal human megakaryocytes as evaluated by semiquantitative RT-PCR. More than 200 megakaryocytic colonies were plucked and pooled on the indicated days of culture, total RNA was extracted, and RT-PCR was performed. The amount of transcripts for PGI2-R or PF4 was analyzed and compared with that of G3PDH. M, size marker (φX174/HaeIII digest). This figure shows the representative results from experiments repeated three times. Yutaka Sasaki et al. Blood 1997;90:1039-1046 ©1997 by American Society of Hematology