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PNAS 105 (21): 7612-7617

Copyright © 2008 by the National Academy of Sciences.

Synergistic upregulation of erythropoietin receptor (EPO-R) expression by sense and antisense EPO-R transcripts in the canine lung

Quiyang Zhang*, Jianning Zhang*, Orson W. Moe*,{dagger},{ddagger}, and Connie C. W. Hsia*,§

Departments of *Internal Medicine and {dagger}Physiology, {ddagger}Charles and Jane Pak Center of Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-9034


Figure 1
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Fig. 1. RNA blot of sense and antisense erythropoietin receptor (sEPO-R and asEPO-R, respectively). (Upper) Location of riboprobes. (Lower) Typical RNA blots labeled with sense or antisense probes under high stringency using either total (20 µg per lane) or polyA+ (1 µg per lane) RNA from lung and total RNA (20 µg per lane) from five other canine organs.

 

Figure 2
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Fig. 2. Expression of sEPO-R and asEPO-R transcripts and EPO-R protein in the remaining canine lung following right PNX or Sham PNX (n = 5 each). (A) RNA blots of sEPO-R and asEPO-R transcripts, with 18S rRNA as loading control. Individual sEPO-R/18S or asEPO-R/18S signals were expressed as a ratio to the mean value in Sham group. Triplicate assays used separate tissue samples from each animal. (B) Immunoblot of EPO-R protein. Mean ± SEM, unpaired t test.

 

Figure 3
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Fig. 3. Schematic diagram of sEPO-R and asEPO-R transcripts from canine lung. The asEPO-R transcript contains two putative ORFs (ORF1 and ORF2, 248 aa each) with hypothetical predicted polypeptides shown (LOC61130 GenBank accession no. XM_84877). Below the native transcripts are cDNA constructs used for expression studies. sEPO-R-FLAG represents sense cDNA with C-terminal FLAG epitope tag. asORF2-long represents antisense cDNA with {approx}300 bp, followed by the full ORF2 coding region. asORF2-short represents antisense cDNA spanning the ORF2 putative coding region. asORF2-shortmut represents asORF2-short with mutated start codon (ATG to ACG) denoted by "X." asORF1 represents antisense cDNA spanning the ORF1 putative coding region. asORF1mut represents asORF1 with mutated start codon (ATG to ACG) denoted by "X."

 

Figure 4
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Fig. 4. Immunolocalization. (A) (Top) sEPO-R (Left) and asEPO-R (Right) transcripts were detected by in situ hybridization (green) using antisense and sense cRNA probes, respectively, in bronchiolar epithelial cells on OCT-embedded lung sections. (Middle) The same sections were labeled for EPO-R protein (red) by immunofluorescence. (Bottom) Superimposed images with differential interference contrast (DIC). (Scale bar, 20 µm.) (B) Immunofluorescent localization of EPO-R protein (red, Center) with an alveolar cell marker (green, Left): AQP5 (Top), SP-C (Middle), and CD34 (Bottom), on the same sections. Images were superimposed with DIC (Right). EPO-R colocalized with SP-C and CD-34 (yellow), but not with AQP5. (Scale bar, 20 µm.)

 

Figure 5
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Fig. 5. Effect of asEPO-R cDNA transfection on EPO-R protein expression in cultured HEK-293 cells. (A) Cells were cotransfected with sEPO-R-FLAG cDNA (2 µg) and variable amounts of asORF2-long cDNA and immunoblotted for EPO-R, FLAG, and β-actin. *, P < 0.05 vs. 0 µg of asEPO-R cDNA. (B) Cells were cotransfected with asORF2-long cDNA (0–2 µg) and variable amounts of sEPO-R-FLAG cDNA and immunoblotted for FLAG and β-actin. *, P < 0.05, 2 µg vs. 0 µg asORF2-long at the same sEPO-R-FLAG cDNA level. For A and B, three independent experiments were performed (mean ± SD, one-way ANOVA). (C) Immunocytochemistry of transfected cells shows increased EPO-R production in response to sEPO-R-FLAG and asORF2-long cotransfection. (Left) Whole-cell images of transfection with vector only, asORF2-long only, sEPO-R-FLAG only, or sEPO-R-FLAG + asORF2-long cDNAs, stained with anti-FLAG antibody for EPO-R-FLAG (red) and phalloidin for β-actin (green). (Scale bars, 20 µm.) (Right) Confocal optical section of cells cotransfected with sEPO-R-FLAG + asORF2-long cDNAs, stained for EPO-R(FLAG) and β-actin. (Scale bar, 10 µm.) (D) Cultured cells were cotransfected with sEPO-R-FLAG cDNA (2 µg) and different antisense cDNAs (2 µg): asORF2-long, antisense cDNA with {approx}300 bp + ORF2 coding region; asORF2-short, antisense cDNA spanning the putative ORF2 coding region; asORF2-shortmut, asORF2-short with mutated start codon; asORF1, antisense cDNA spanning the putative ORF1 coding region; asORF1mut, asORF1 with mutated start codon. EPO-R-FLAG expression was detected by immunoblot with anti-FLAG normalized to β-actin. A typical blot (Upper) and average data from four independent experiments (Lower) are shown. The mean EPO-R-FLAG/β-actin expression with vector only was arbitrarily set at 100%. Mean ± SD, one-way ANOVA: P < 0.05 * vs. empty vector {dagger} vs. corresponding wild-type antisense cDNA.

 


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