Novel Cis Element for Tissue-Specific Transcription of Rat Platelet-Derived Growth Factor β-Receptor Gene by Yasunori Takata, Yutaka Kitami, Tomikazu Fukuoka, Takafumi Okura, and Kunio Hiwada Hypertension Volume 33(1):298-302 January 1, 1999 Copyright © American Heart Association, Inc. All rights reserved.

Northern blot analysis of PDGFβR mRNA in VSMC, pulmonary fibroblasts, and HTC. Total cellular RNA (10 μg) obtained from quiescent VSMC (lane 1), pulmonary fibroblasts (lane 2), and HTC (lane 3) were analyzed by Northern blotting for PDGFβR mRNA (5.6 kb). Northern blot analysis of PDGFβR mRNA in VSMC, pulmonary fibroblasts, and HTC. Total cellular RNA (10 μg) obtained from quiescent VSMC (lane 1), pulmonary fibroblasts (lane 2), and HTC (lane 3) were analyzed by Northern blotting for PDGFβR mRNA (5.6 kb). Lower panel indicates ethidium bromide staining of 28S and 18S rRNA. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.

Plasmid constructs of promoter-luciferase fusion genes and their luciferase activities in VSMC or HTC. Luc-1,681 to Luc-120 indicate progressive 5′-deletions, which had nucleotide sequences starting at the positions, −1,681, −310, −271, −230, −190, −150, and −120, respectively. pGLB indicates a promoterless luciferase vector. Plasmid constructs of promoter-luciferase fusion genes and their luciferase activities in VSMC or HTC. Luc-1,681 to Luc-120 indicate progressive 5′-deletions, which had nucleotide sequences starting at the positions, −1,681, −310, −271, −230, −190, −150, and −120, respectively. pGLB indicates a promoterless luciferase vector. Each construct was transiently cotransfected with pRL-CMV into VSMC (closed columns) or HTC (open columns) with the method of DEAE-dextran. Promoter activity was assessed in terms of its ability to drive firefly-luciferase cDNA expression after this had been normalized with respect to renilla-luciferase cDNA expression and was finally presented as relative luciferase activity with reference to the activity of Luc-1,681 which was set to 100%. *P<0.05, significant difference compared with Luc-1,681; †P<0.05, significant difference between VSMC and HTC. Data are expressed as mean+SEM of 4 separate assays. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.

EMSA for CCAAT motif located at −67 and the R30 spanning −150 to −121 with nuclear extracts from VSMC. End-labeled probe for C67 (lane 1 to 3) or R30 (lane 4 to 6) was incubated with 2 μg of nuclear extracts from quiescent VSMC. For competition experiment, 100-molar excess of unlabeled C67 (lanes 2 and 6) or unlabeled R30 (lanes 3 and 5) was incubated with nuclear extracts for 15 minutes before adding labeled probe. EMSA for CCAAT motif located at −67 and the R30 spanning −150 to −121 with nuclear extracts from VSMC. End-labeled probe for C67 (lane 1 to 3) or R30 (lane 4 to 6) was incubated with 2 μg of nuclear extracts from quiescent VSMC. For competition experiment, 100-molar excess of unlabeled C67 (lanes 2 and 6) or unlabeled R30 (lanes 3 and 5) was incubated with nuclear extracts for 15 minutes before adding labeled probe. Lanes 1 and 4 indicated no competitor. Positions of specific DNA-protein complex are indicated as B1-B3; free DNA probe as Free. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.

Supershift assay for CCAAT motif located at −67 with antibodies against NF-Y family. Supershift assay for CCAAT motif located at −67 with antibodies against NF-Y family. Labeled C67 probe was added to nuclear extracts (2 μg) from quiescent VSMC and was incubated with 1 μL of specific antibodies against NF-YA (A), NF-YB (B), and NF-YC (C) or preimmune serum (PI) for 15 minutes before electrophoresis. Positions of specific protein complexes were indicated as B1 and B2; supershifted bands as asterisks. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.

Effect of cis element decoy ODN against C67 or R30 on endogenous PDGFβR mRNA expression. Effect of cis element decoy ODN against C67 or R30 on endogenous PDGFβR mRNA expression. Phosphorothioate double-stranded ODN was prepared for C67 (lane 2) or R30 (lane 3) as cis element decoy. In addition, scramble decoy ODN (lane 1) was also prepared for C67 as control. Each decoy ODN was mixed with Lipofectamine Plus and was directly added to culture medium at final concentration of 1 μmol/L. After incubation for 3 hours, cells were washed with PBS and cultured for 24 hours before isolation of RNA. Northern blot analysis for PDGFβR mRNA was performed by same method described in Figure 1. Amounts of endogenous PDGFβR mRNA were presented as relative expression levels with reference to level of control scramble decoy, which was set to 100%. Data are expressed as mean+SEM of 4 separate assays. †P<0.05, significant difference compared with level of control scramble decoy. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.

Comparison of EMSA pattern between VSMC and HTC Comparison of EMSA pattern between VSMC and HTC. EMSA pattern for C67 or R30 was compared between nuclear extracts from VSMC (lanes 2 and 4) and HTC (lanes 1 and 3). Comparison of EMSA pattern between VSMC and HTC. EMSA pattern for C67 or R30 was compared between nuclear extracts from VSMC (lanes 2 and 4) and HTC (lanes 1 and 3). Nuclear extracts (2 μg) were incubated with labeled C67 probe (lanes 1 and 2) or labeled R30 probe (lanes 3 and 4) under same conditions as described in Figure 3. Positions of specific DNA-protein complex observed in VSMC are indicated as B1 and B2; free DNA probe as Free. Closely shifted band (*) was specifically observed in HTC (lane 3) in addition to B3. Yasunori Takata et al. Hypertension. 1999;33:298-302 Copyright © American Heart Association, Inc. All rights reserved.