Figure 3
From: Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1
Characterization of MC3T3-E1 cells over-expressing hPiT1 or hPiT2. A) qRT-PCR analysis of exogenously expressed hPiT1 and hPiT2 mRNA levels in MC3T3-E1-LXSN (LXSN), MC3T3-E1-LPiT1SN (LPiT1SN), and MC3T3-E1-LPiT2SN (LPiT2SN) cells, respectively. Each column represents cell lysates from three wells and triplicate qRT-PCR analyses of each cell lysate. The hPiT1 and hPiT2 mRNA levels are standardized to the endogenous B2M mRNA levels. Data are means ± SD. "No CT" indicates that no signal was obtained for the transgene examined. * indicates statistically significantly different from MC3T3-E1-LXSN (control) cells, p <0.05. B) RBD-binding assay. MC3T3-E1-LXSN (LXSN), MC3T3-E1-LPiT1SN (LPiT1SN), and MC3T3-E1-LPiT2SN (LPiT2SN) cells were incubated with His-tagged FeLV-B RBD or His-tagged A-MLV RBD containing supernatant (dashed line) or in standard growth medium containing no RBD (solid line) followed by a mouse-anti-His antibody and finally a PE-conjugated goat anti-mouse Ig antibody. The cells were analyzed by flow cytometry. The x-axis shows PE intensity measured in the FL2-H channel. C and D) Expression of endogenous mPiT1 and mPiT2. MC3T3-E1-LXSN (LXSN), MC3T3-E1-LPiT1SN (LPiT1SN), and MC3T3-E1-LPiT2SN (LPiT2SN) cells were seeded at 500 cells/cm2 in 6-well plates and the endogenous mPiT1 mRNA levels were analyzed by qRT-PCR at the indicated days in culture. Each column represents six wells, but since the wells were harvested two and two in the same cell lysis buffers, the columns represent three lysates, and triplicate qRT-PCR analyses of each cell lysate. The mPiT1 and mPiT2 mRNA levels are standardized to B2M mRNA levels. Data are means ± SD. E) Pi-transport assay. MC3T3-E1-LXSN (LXSN), MC3T3-E1-LPiT1SN (LPiT1SN), and MC3T3-E1-LPiT2SN (LPiT2SN) cells were seeded at 20,000 cells/cm2 in 4-well plates. The next day, 32Pi import in medium containing a total [Pi] of 100 μM was analyzed over 5 minutes. The results are shown as mean 32Pi import per mg protein per hour of 4 wells ± SD. * indicates statistically significantly different from MC3T3-E1-LXSN (control) cells, p < 0.05.