B6Tert 1 cells exhibit the characteristics of normal EVTs by producing various

Analysis of the mechanism of receptor tyrosine kinase activation in monocytes may identify soluble factors that control PCMO self renewal. The present study aimed to investigate the expression and the activity of the epidermal growth factor receptor family in human peripheral monocytes and the role of EGF and HB EGF on the outcome INNO-406 SRC 阻害剤 of PCMO generation and the subsequent differentiation into NeoHepatocytes. Results Gene expression of EGF receptor family members in PCMOs We first sought to determine which EGF receptors are expressed in monocytes. For this purpose, RNA was iso lated from monocyte cultures and processed for qPCR using primers for EGFR, ERBB2, ERBB3, and ERBB4 as listed in Table 1. RT PCR analysis of the four EGF receptors yielded a strong signal for EGFR and a weaker one for ERBB3.<br><br> Since monocytes Lapatinib 388082-77-7 may be contaminated with lymphocytes, a negative control sample of highly purified lymphocytes was analyzed in parallel and shown to lack expression of both EGFR and ERBB3. This indicated that the amplification products for EGFR and ERBB3 were specifically derived from monocytes. Since the ex pression levels of some genes may differ during the de velopment of PCMOs in culture, we isolated RNA from the developing PCMOs at different days of culture. The qPCR of these samples indicated that expression of both EGFR and ERBB3 initially increased during PCMO gen eration reaching a peak on the second day and on the fourth day of culture and decreased thereafter. EGF promotes proliferation during PCMO production Next, we examined the effect of EGF and HB EGF on the proliferation of PCMOs.<br><br> For this purpose, cells were cultured supplier Lonafarnib for 4 days in PCMO medium con taining EGF or HB EGF at different concentrations. Cells were prepared for immunofluorescence using Ki67 antibody as a proliferation marker and CD14 as a mono cyte marker. The results showed a higher number of Ki67CD14 double positive cells in both EGF and HB EGF treated cultures. However, quantifica tion of these cells showed that the HB EGF but not the EGF effect closely missed statistical significance. No statistically significant differences of Ki67CD14 positive cell counts were observed among different concentrations of the same treatment. These data clearly show that the addition of EGF enhanced the proliferative activity of monocytes in PCMO generation medium.<br><br> EGF induced proliferation temporally corre lated with cell cycle activation. In order to investigate whether EGF induced proliferation was associated with the expression of specific cell cycle regulatory genes, we treated monocytes with different concentrations of EGF or HB EGF and performed qPCR analysis as described in the Methods section. As seen in Table 2, both EGF and HB EGF up regulated the expression of ABL, ANAPC2, CDC2, CDK4, and CDK6, each of which is involved in different stages of the cell cycle. RNA was isolated from PCMOs after 4 day culture with or without EGF or HB EGF and tran scribed to cDNA. QPCR was applied using primer pairs listed in Table 1. Data are presented as mean SEM of N 4 and represent the fold change in comparison with control PCMOs, the values of which were considered as 1.