CD68+ and F4/80+ macrophages are analogous populations in PyMT tumors.

<p>Tumors from PyMT mice were enzymatically digested and made into a single cell suspension. Cells were stained with antibodies against the surface markers CD45, CD11b and F4/80. Cells were then fixed, permeabilized and stained with anti-CD68. Fluorescence was measured by flow cytometry and the data was analyzed using Flowjo software. Dot plots shown were generated from CD45⁺ cells. CD68 expression was determined after gating on tumor-associated macrophages (CD45⁺CD11b⁺F4/80⁺), CD11b⁺F4/80^—cells and CD11b^—F4/80^—cells. Representative dot plots are shown from one of four mice analysed.</p

Doxycycline reduces the number of macrophages surrounding mammary tumors in PyMT-MacLow mice.

<p>(<b>A</b>) Sections of mammary tissue were labelled for F4/80 (DAB brown cells) and counterstained with haematoxylin; a representative image at the early carcinoma stage of tumor development is shown for each genotype and treatment. (<b>B</b>) Images captured from slides scanned on an Aperio slide scanner were used to quantify the number of macrophages within (intratumoral) and on the perimeter of mammary tumors (peritumoral). The data was then grouped according to genotype and treatment group and the mean value +/- SD shown. Each individual data point represent the mean values for an individual tumor. Tumor samples were obtained from the following numbers of animals per treatment group the number of tumors analysed is indicated in brackets: PyMT UT = 5(27), PyMT Doxy = 5(33), PyMT-MacLow UT = 7(31), PyMT-MacLow Doxy = 7(39). Significance is from the SPSS nested analysis comparing data from doxycycline treated versus control animals for each tumor and genotype. NS = not significant, **P<0.01. Scalebar = 100 μm. (C) The percentage of animals that had any tumors of hyperplasia and/or Adenoma/mammary intraepithelial neoplasia stages (Adenoma/MIN) was calculated for each genotype and treatment group. A Chi-square test was used to calculate statistical significance.</p

A general RT-PCR approach using primers to exon 3 or 4 and 3'UTR failed to detect VEGFxxxb isoforms in mouse and human cDNA extracts respectively.

<p><b>A</b>, PCR products corresponding to VEGF188 (474 bp), VEGF164 (402 bp), VEGF164/120 heteroduplex (arrowed) and VEGF120 (270 bp) are evident in the panel of mouse cDNAs amplified using the 3′UTR reverse primer and a forward primer to exon 3. <b>B</b> & <b>C</b>, PCR products corresponding to VEGF189 (371 bp), VEGF165 (299 bp) and VEGF121 (167 bp) are evident in the commercial human tissue cDNAs amplified using the 3′UTR reverse primer (DB 3'UTR) and a forward primer to exon 4 (DB exon4). <b>D</b>, Amplification of the heteroduplex species (arrowed) a lso occurred when VEGF164 and VEGF120 tumour cDNAs were pooled and analysed by RT-PCR using exon3/3'UTR primers (lanes labelled 120+164). <b>E</b>, The same heteroduplex species was generated when cDNAs from VEGF164 and VEGF188 expressing fibrosarcoma cells were pooled and amplified as above (lanes labelled 120+164). M corresponds to a 100 bp ladder, whilst -tem represents a control PCR reaction in which water was used instead of cDNA template. The Figure contains data we published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035231#pone.0035231-Tozer1" target="_blank">[25]</a>.</p

The proliferative capacity of Adenoma/MIN tumors is reduced in macrophage deficient mice.

<p>Mammary sections were labelled with an anti-Ki67 antibody as a marker of proliferation and counterstained with haematoxylin. (<b>A</b>) Images were captured from slides scanned on an Aperio slide scanner and split according to tumor grade, a representative image of an Adeno/MIN tumor (A/M) is shown for each genotype and treatment group. (<b>B</b>) The amount of Ki67 labelling in individual tumor areas (cells stained dark brown with DAB) was quantified and expressed as positivity using the Aperio positive pixel algorithm. Each data point on the graph represents the mean positivity for an individual tumor and the number of animals these tumors were taken from is shown below the x axis on each graph. The mean value ± SD was plotted and a nested analysis carried out in SPSS to compare data from doxycycline treated versus control animals from each tumor grade and genotype. ***P<0.001. Scalebar = 100 μm.</p

Effect of IGF-1 and TGFβ-1 on expression of VEGF isoforms in mouse podocytes.

<p>Cell lysates were prepared from untreated control (C) podocyte cells or cells treated with either 100 nM IGF-1 (IGF) or 1 nM TGFβ-1 (TGF) for 12 hours in serum free media. Results from three independent experiments are shown. RT-PCR using general primers designed to simultaneously amplify both VEGFxxx and VEGFxxxb isoforms (exon7a/3'UTR) revealed only VEGFxxx (194 bp). Qualitative assessment of the PCR products suggests that treatment with either growth factor increased VEGFxxx expression. The same RT-PCR strategy using three different extracts (1, 2, 3) from HEK 293 cells similarly revealed only VEGFxxx (194 bp). (-tem) corresponds to reactions containing water instead of cDNA. (-RT) corresponds to reactions containing water instead of reverse transcriptase.</p

A general RT-PCR approach with primers to exon 7a and 3'UTR failed to detect VEGFxxxb isoforms in human and mouse cDNA extracts.

<p>RT-PCR reactions were performed using forward/reverse primers designed to amplify both VEGFxxx and VEGFxxxb isoforms from cDNAs extracted from mouse fibrosarcoma cell lines & tumours and normal mouse tissues (heart, lung, liver and kidney) as well as commercially sourced human tissue cDNAs. <b>A</b>, A single product corresponding to VEGF164/188 (194 bp) is evident in the panel of mouse cDNAs using forward and reverse primers designed to exon 7 and the 3'UTR (exon7/3′UTR) with no evidence of VEGF164b/188b (128 bp) products. <b>B</b> & <b>C</b>, A single product corresponding to VEGF165/189 (194 bp) is evident in human brain, bladder and kidney (AMS Biotechnology Ltd), and prostate and kidney (Primer Design Ltd) using forward and reverse primers designed to exon 7 (DB exon7a) and the 3'UTR (DB 3'UTR) respectively. No products corresponding to VEGF165b/189b (128 bp) are evident in any of these samples.</p

Murine VEGF-A splice variants.

<p>Several splice variants of VEGF are produced, by alternative splicing of exons 6 & 7. Exons 3 & 4 encode dimerisation and VEGF receptor binding sites, exon 7 encodes neuropilin binding sites and exons 6 & 7 encode heparin binding sites.</p

Discriminating VEGF isoform-specific RT-PCR using human cDNA extracts further highlights the importance of primer design.

<p><b>A</b>, Human cDNAs from colon, prostate and kidney (Primer Design Ltd) were amplified in RT-PCR reactions using an exon 4 forward primer together with either DB165b/189b-1 (lane 1), h165b/189b-2 (lane 2), h165b/189b-3 (lane 3), h165b/189b-4 (lane 4) or h165b/189b-5 (lane 5). Primer sequences are highlighted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035231#pone-0035231-t001" target="_blank">Table 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035231#pone-0035231-g002" target="_blank">Fig 2</a>. A putative VEGF165b (∼212 bp) product is evident in lanes 2 & 3, but absent in lane 1. All three reverse primers spanned 5 bases across the 8b/7 junction, however differed in their melting temperatures (see main text), with h165b/189b-2 & 3 exhibiting more compatible Tms with the forward primer. A similar 165b product was more abundantly amplified in lanes 4 & 5 using reverse primers spanning more bases (13 or 14 respectively) across the 8b/7 splice junction. In addition, these latter reverse primers also amplified a product corresponding to 188b (∼283 bp). However DNA sequencing revealed that the product amplified in lanes 2 & 3 was not VEGF but LIM domain only, protein isoform 7, confirming that detection of apparent VEGFxxxb species was only evident using 8b/7 reverse primers with increasing complementary sequence to exon7. <b>B</b>, Similar results were obtained in RT-PCR reactions using cDNA extracted from a normal kidney patient biopsy and amplified with the exon 4 forward primer and the DB165b/189b-1 (lane1), h165b/189b-2 (lane 2) and h165b/189b-4 (lane 4) reverse primers. <b>C</b>, RT-PCR using the DBexon7/DB3'UTR forward/reverse primers capable of simultaneously amplifying VEGFxxx & VEGFxxxb, confirmed only VEGFxxx (194 bp) amplification in the human samples tested.</p

Mouse and human PCR primer sequences.

<p>The sequences of all forward and reverse PCR primers used in this study are highlighted along with their corresponding melting temperatures. The junction spanning details of the VEGFxxxb isoform-specific primers are discussed in the main text (highlighted in bold above).</p

C-terminal exon sequences of murine and human VEGF-A genes.

<p>Top two panels show the predicted splicing reactions for murine VEGF188b, 164b and 120b (sequence in bold and arrowed), highlighting the exon 7/exon 8b and exon 5/exon 8b splice junction sequences. These splicing reactions would generate putative VEGFxxxb protein isoforms with a <b><i>PLTGKTD</i></b> C-terminus. The reverse PCR primers designed for this study are indicated below the exon sequences. The lower panel shows the corresponding sequence of the human VEGF-A gene C-terminus highlighting the exon 7/exon 8b splice junction that generates VEGFxxxb isoforms with a <b><i>SLTRKD</i></b> C-terminus. The reverse PCR primers used in the amplification reactions are indicated below the exon sequences and DB 165b/188b-1 and DB 3'UTR are as previously published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035231#pone.0035231-Bates1" target="_blank">[26]</a>. The bases highlighted in bold exhibit mismatches from the published human VEGF-A sequence.</p