PKC plays a crucial roles in c-mpl gene expression in megakaryoblastic cells

Abstract

Summary: Thrombopoietin is the cytokine involved in megakaryopoiesis and its receptor (c-[Short communication]

GATA-dependent regulation of TPO-induced c-mpl Gene Expression during Megakaryopoiesis

“…As described previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) , 2008 16) ), the cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% FCS, 2mM L-glutamine, 100 units/ml penicillin, 100µg/ ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in humid air containing 5% CO 2 atmosphere. CMK cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 100 units/ml penicillin, 100 µg/ ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in humidified air containing 5% CO 2 atmosphere.…”

“…The construct [pGL3-c-mpl (-310)] has been reported previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) ), the 310 bp upstream promoter region of c-mpl was amplified from the chromosomal DNA of the CMK cells by polymerase chain reaction using the following primer set, (forward 5'-CTA TTC TGA TGC TAT TAT ACC ATT C-3', reverse 5'-CTT GGC TGC TGA CTT GGG C-3'). The PCR product was cloned into pGEMTeasy vector (Promega, Madison, WI) and designated as pGEM-c-mpl-prom.…”

“…As described previously (Sunohara et al, 2003 11) , 2013 13) , 2009 14) , 2010 15) ), three reporter plasmids, pGL3-basic vector (as a negative control), pGL3-control vector in which the luciferase gene is driven under SV40 promoter (as a positive control, Promega) and the pGL3-c-mpl (-310) were used for transfection assay. A plasmid, Fig.…”

Role of promoter element in c-mpl gene expression induced by TPO

“…As described previously (Sunohara et al, 2003 11) , 2009 13) , 2010 14) , 2008 15) ), the cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) containing 10% FCS, 2mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37°C in humid air containing 5% CO 2 atmosphere. CMK cells were cultured in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B at 37 °C in humidified air containing 5% CO 2 atmosphere.…”

“…As described previously (Sunohara et al, 2003 11) , 2009 13) , 2010 14) ), three reporter plasmids, pGL3-basic vector (as a negative control), pGL3-control vector in which the luciferase gene is driven under SV40 promoter (as a positive control, Promega) and the pGL3-c-mpl (-310) were used for transfection assay. A plasmid, pRL-CMV (TOYO INC MFG CO., LTD., Tokyo), which expresses Renilla (SeaPansy) luciferase under control of CMV early promoter, was used to normalize the transfection efficiency.…”