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. Author manuscript; available in PMC: 2009 Nov 25.
Published in final edited form as: Nat Cell Biol. 2009 Apr 12;11(5):604–615. doi: 10.1038/ncb1866

Figure 2. IGF-1R and GHR are repressed upon persistent DNA damage and leads to IGF-1 resistance.

Figure 2

A) Three independent primary mouse dermal fibroblast cell lines (WT1–3; n=3)) at early passage were treated with UV (UV-C, 254 nm) and RNA samples were taken 6 hrs post treatment. IGF-1R and GHR qPCRs were done in duplicate and normalized to gamma-tubulin (gTUB) and beta-2-microglobulin (B2M). B) Primary chondrocytes were treated similarly and IGF-1R and GHR expression levels normalized to B2M, gTUB, and Hprt (average of two independent chondrocyte lines shown) and (C) IGF-1R and GHR protein and phosphorylation levels of STAT5 and AKT were quantified 12 h after UV treatment using fluorescently labeled antibodies and normalized to Actin levels (left panels: representative experiment; right panel: average of three independent experiments, n=3). D) Two independent human fibroblast lines (C5RO and C3RO) were treated with indicated doses of UV. IGF-1R and GHR expression levels were determined 6 hrs post treatment and normalized to Gapdh and B2M. E) IGF-1R down-regulation is sufficient to reduce cell growth. U2OS cells were counted 96 hours after transfection with either non-targeting siRNA oligos or siRNA oligos targeting IGF-1R (daily division rate given, n=4; upper right panel IGF-1R protein levels, lower right panel IGF-1R mRNA levels (qPCR)). F) IGF-1R ablation antagonizes hyperplasia. 8 – 10 weeks old Igf-1repi−/− and control mice were shaved and after 24 hours, treated three times every 48 hours with 10nm TPA/330µl or solvent alone (aceton). Upper panel: H&E section showing epidermal hyperplasia following TPA treatments. Lower panel: Quantification of hyperproliferative response (TPA-treated: n = 3–5, aceton treated: n = 2; two-tailed t-test). UV induces IGF-1 resistance: (G) U2OS cells were serum starved for 2 hours starting at 12 hours after UV treatment and treated with 100ng/mL IGF1 for 10 minutes. H) Medium was supplemented with IGF-1 after UV treatment and cells were counted 48 hours after treatment. (FC = fold change compared to untreated controls; *=p<0.05, **=p<0.01, two-tailed t-test, error bars = SD, n=4)