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Dunlap et al. 10.1073/pnas.0703145104. |
Supporting Information
Files in this Data Supplement:
SI Figure 4
SI Figure 5
SI Figure 6
SI Figure 4Fig. 4. GFP negative control injections result in isolated inflammation and no tumor. H&E stain shows the histological characteristics of isolated cases of inflammation in GFP control injections of N-tva mice. (A) GFP injections can result in focal intraparenchymal inflammatory cell infiltrates (arrow). (B) GFP injections can also result in chronic inflammatory cell (lymphocytic) infiltrates in the subarachnoid space (arrow). (C) GFP injections can result in acute inflammatory cell (polymorphonuclear leukocyte) infiltrates (arrows). (D) Whole-mount photomicrograph showing hydrocephalus and associated encephalomalacia present in <10% of control-injected mice (arrows). No tumors formed in GFP control-injected mice, as expected. D is ´1.5 original magnification.
SI Figure 5Fig. 5. Immunohistochemistry of PDGFB and IGFBP2 injections. Cell morphology, injection markers, reactive astrocytes, and cell proliferation are shown. (A and B) H&E stains show typical oligodendroglioma histological features in PDGFB-driven tumor (A) and increased cellular density with microvascular proliferation in PDGFB-IGFBP2-driven anaplastic oligodendroglioma (arrows) (B). (C and D) Immunostain with anti-HA tag antibody shows broad expression of PDGFB in both tumor sets (arrows). The RCAS-PDGFB injected in this study contained an HA tag. (E and F) Immunostain with anti-GFP antibody also shows broad expression of GFP in both tumor sets (arrows). GFP was coinjected in every injection set as an injection marker. GFP was also injected alone and formed no tumors. (G and H) Anti-GFAP immunostain labels reactive astrocytes within the oligodendrogliomas (arrows). This pattern of reactive astrocytosis recapitulates that seen in human oligodendrogliomas.
SI Figure 6Fig. 6. IGFBP2-coinfected cells show up-regulation and activation of the Akt pathway. Brains from 1-day-old N-tva mice were harvested, and N-tva-positive primary cells were cultured in 10% FBS, DMEM high-glucose. Primary cultures were then treated with filtered, conditioned media from RCAS-infected DF-1 cells once per day for 3 weeks. Infected primary cultures were then harvested and used for Western blot analysis. Lane 1 shows noninfected cells, which were not incubated with any conditioned media. Lane 2 shows cells that were incubated only with GFP constructs, which served as a visible infection marker. Lane 3 shows cells incubated with PDGFB and GFP. Lane 4 shows cells incubated with PDGFB, IGFBP2, and GFP. Coinfection with IGFBP2 did not change the levels of total PI3K. However, an up-regulation of total Akt as well as an increase in pAkt(Thr308) and pAkt(Ser473) in IGFBP2 coinfections was seen. An increase in the levels of total S6K, as well as an increase in p-S6K, in IGFBP2 coinfections was also seen. GFP served as a visible infection marker as well as an infection control on the Western blot.
