American Society of Hematology

Figure 1.

$Figure 1. TPO controls VEGF-A at a transcriptional level. (A) To analyze the expression of VEGF-A variant forms, RNA was prepared from UT-7/TPO cells cultured with 10 ng/mL hTPO and RT-PCR was performed with primers spanning exon 3 and exon 8. (B) UT-7/TPO cells were deprived of growth factors for 24 hours (□) and then stimulated with 100 ng/mL hTPO for various time periods (▨, 1 hour; ▦, 6 hours; ▪, 24 hours). RNA was prepared and VEGF-A mRNA levels were analyzed by real-time RT-PCR with primers that recognize all variants of VEGF-A transcripts. Each column represents the average VEGF-A level normalized to a GAPDH internal control ± SD of 3 independent experiments. (C) After 24 hours growth factor deprivation, UT-7/TPO cells were stimulated with 100 ng/mL hTPO for 3 hours, after which 10 μg/mL actinomycin D was added and the cells cultured for the additional indicated time periods with (○ and solid line) or without (▪ and broken line) hTPO. RNA was prepared and VEGF-A levels were monitored by the real-time RT-PCR assay. Levels of 18s RNA were used as an internal control. The graph represents the result of 3 independent experiments ± SD. The difference between the 2 curves does not reach statistical significance. (D) UT-7/TPO cells were deprived of growth factors for 24 hours and then treated with 100 ng/mL hTPO. The cytoplasmic protein fraction was prepared and VEGF-A expression levels were analyzed by Western blotting. *VEGF165; **VEGF121.$

TPO controls VEGF-A at a transcriptional level. (A) To analyze the expression of VEGF-A variant forms, RNA was prepared from UT-7/TPO cells cultured with 10 ng/mL hTPO and RT-PCR was performed with primers spanning exon 3 and exon 8. (B) UT-7/TPO cells were deprived of growth factors for 24 hours (□) and then stimulated with 100 ng/mL hTPO for various time periods (▨, 1 hour; ▦, 6 hours; ▪, 24 hours). RNA was prepared and VEGF-A mRNA levels were analyzed by real-time RT-PCR with primers that recognize all variants of VEGF-A transcripts. Each column represents the average VEGF-A level normalized to a GAPDH internal control ± SD of 3 independent experiments. (C) After 24 hours growth factor deprivation, UT-7/TPO cells were stimulated with 100 ng/mL hTPO for 3 hours, after which 10 μg/mL actinomycin D was added and the cells cultured for the additional indicated time periods with (○ and solid line) or without (▪ and broken line) hTPO. RNA was prepared and VEGF-A levels were monitored by the real-time RT-PCR assay. Levels of 18s RNA were used as an internal control. The graph represents the result of 3 independent experiments ± SD. The difference between the 2 curves does not reach statistical significance. (D) UT-7/TPO cells were deprived of growth factors for 24 hours and then treated with 100 ng/mL hTPO. The cytoplasmic protein fraction was prepared and VEGF-A expression levels were analyzed by Western blotting. ^*VEGF165; ^**VEGF121.

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