AT2 receptor interacting protein 1 (ATIP1) mediates COX-2 induction by an AT2 receptor agonist in endothelial cells

Figures

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  Fig. 1. Immunofluorescence analysis of ATIP1 localization in HUVECs using the anti-ATIP monoclonal antibody C1717. (A) C1717 detected a single 50 kDa band in HUVEC lysate. (IB; immunoblot) (B) HUVECs were stained with C1717, an anti-VE-cadherin antibody (Ab) (Upper panels) and C1717, an anti-golgin 97 Ab (Lower panels). Co-localization of ATIP1 and VE-cadherin (right upper panel) and golgin97 (right bottom panel) are shown in yellow. Scale Bar; 20 μm. (C) The C1717 reactive bands are diminished in siATIP-C treated HUVEC lysate compared to siControl-C. (D) HUVECs were transfected with siControl or siATIP for 48 h. The siControl-N and siATIP-N treated HUVECs were stained with C1717 and MitoTracker. The majority of ATIP1 co-localized to mitochondria, as shown in the upper right panel, an effect which disappeared in HUVECs when ATIP1 was knocked down (right bottom panel). Scale Bar; 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 2. qPCR analysis in HUVECs of COX-2 mRNA induction by CGP42112A or Ang II stimulation. (A), (B) CGP42112A (10−8 M) upregulated COX-2 mRNA significantly under siControl-N (A) or siControl-C (B) compared to siATIP-N or siATIP-C, respectively (ANOVA, *p < 0.05). The significance between each treatment on the Post Hoc test at the indicated hour is shown (*p < 0.05). (C) Ang II (10−7 M) upregulated COX-2 mRNA, which was suppressed by knocking down siATIP-C (*p < 0.05). Values are the mean ± SEM for all three experiments.

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  Fig. 3. An AT2R specific inhibitor suppressed COX-2 mRNA induction by Ang II stimulation in HUVECs. (A) PD123319 (10−5 M) treatment completely suppressed the COX-2 mRNA induction by Ang II (10−7 M) in HUVECs. (B) Treatment with the AT1R blocker telmisartan (10−5 M) suppress Ang II (10-7 M) or CGP42112A (10-8 M) upregulation of COX-2 mRNA in HUVECs. Values are the mean ± SEM for all three experiments. *p < 0.05.

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  Supplemental Fig. 1. ATIP1 variant expression in HUVECs and anti-ATIP monoclonal antibody C1717 generation. (A) The structural organization of the MTUS1 gene is quoted from the work of Di Benedetto et al. [20]. We designed primers in alternative exons used by the ATIP transcript variants as shown in Material and Methods. siATIP-N in exon 8 and siATIP-C in exon 17 indicates the siRNA region used to knockdown ATIP. (B) The Primer pairs, size (bp) and predicted ATIP transcript variants are listed. (C) The RT-PCR using the primers shown in (B) showed that the ATIP1 variant was expressed in HUVECs but not HeLa cells. (D) We selected the site of the immunogen to generate the anti-ATIP monoclonal antibody in the C-terminal region of mouse ATIP1 (327-426 a.a.), as indicated by both ends of the arrow. (E) C1717 detected Myc-tagged human ATIP1 transiently overexpressed in Hela cells as two bands of approximately 50 kDa. Overexpressed ATIP1 was also detected by anti-Myc antibody at the same size when using C1717. IB; immunoblot. Vehicle; cell lysate of pcDNA4 plasmid transfected to Hela cells. (F), (G) qPCR showed that endogenous ATIP1 was knocked down with siATIP-N or siATIP-C at the mRNA level. Values are the…

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  Supplemental Fig. 2. AT2R and AT1R are expressed in HUVECs. (A) We selected the site of the primers to amplify the C-terminal region of AT2R (Probe 1 and Probe 2) and AT1R (Probe 3 and Probe 4) as indicated by arrows. (B) The Primer pair and predicted PCR-product size (bp) are listed. (C) Amplified PCR products run on 1.5% agarose gel electrophoresis showed that the AT2R and AT1R were expressed in HUVECs.