M-CSF regulates VEGF mRNA transcription through ERK and Sp1.

<p>A) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours in minimal media. The cells were stimulated with 100 ng/ml rhM-CSF for 6 hours followed by isolation of RNA. cDNA was synthesized from total cellular RNA, standardized, and subjected to SYBR Green real-time PCR using primers specific for VEGF-A. The data represents the mean+/−SEM of six individual donors. B) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hrs followed by pre-treatment with either DMSO (vehicle control) or U0126 (10 µM) in minimal media for 30 minutes. The cells were then stimulated with 100 ng/ml rhM-CSF <i>(M-CSF)</i>, <i>(M-CSF+DMSO)</i>, <i>(M-CSF+U0126 10 µM)</i> and analyzed as stated in A. The data represents the mean+/−SEM of three individual donors. C) Freshly isolated human monocytes were cultured overnight in 5% FBS and subsequently starved for 2 hours followed by pre-treatment with either methanol (vehicle control) or mithramycin (200 nM) in minimal media. The cells were then stimulated with 100 ng/ml rhM-CSF <i>(M-CSF)</i>, <i>(M-CSF+MeOH)</i>, <i>(M-CSF+Mith (200 nM))</i> and analyzed as in A. The data represents the mean+/−SEM of four individual donors.