- Fujita Y.
- Sakakura C.
- Shimomura K.
- Nakanishi M.
- Yasuoka R.
- Aragane H.
- Hagiwara A.
- Abe T.
- Inazawa J.
- Yamagishi H.
- Tilli M.T.
- Reiter R.
- Oh A.S.
- Henke R.T.
- McDonnell K.
- Gallicano G.I.
- Furth P.A.
- Riegel A.T.
Materials and Methods
Short Hairpin RNA Constructs and Lentivirus Infection
- Moffat J.
- Grueneberg D.A.
- Yang X.
- Kim S.Y.
- Kloepfer A.M.
- Hinkle G.
- Piqani B.
- Eisenhaure T.M.
- Luo B.
- Grenier J.K.
- Carpenter A.E.
- Foo S.Y.
- Stewart S.A.
- Stockwell B.R.
- Hacohen N.
- Hahn W.C.
- Lander E.S.
- Sabatini D.M.
- Root D.E.
Tube Formation Assay
Monolayer Formation Assay
Cell Migration Assay
Studies in Animals
Matrigel Plug Assay
Quantitative Real-Time PCR
|Gene||Forward primer||Reverse primer|
In Vivo Fluorescence Imaging
Western Blot Analysis
Data Analysis and Statistics
AIB1/SRC-3 Depletion Impairs Endothelial Cell Function in Vitro
Role of AIB1/SRC-3 for Angiogenesis in Vivo
Role of AIB1/SRC-3 in the Healing of Full-Thickness Skin Wounds
Contribution of AIB1/SRC-3 to the Inflammatory and Angiogenic Responses during Wound Healing
Driver Pathways of AIB1/SRC-3 Effects during Wound Healing
- Fujita Y.
- Sakakura C.
- Shimomura K.
- Nakanishi M.
- Yasuoka R.
- Aragane H.
- Hagiwara A.
- Abe T.
- Inazawa J.
- Yamagishi H.
- Supplemental Figure S1
AIB1 mRNA expression in the stroma of human breast cancer relative to normal breast stroma. Analysis of two published expression arrays of stromal tissues20,21obtained from the Oncomine database (http://www.oncomine.org). Values are given as the mean ± SEM and are shown on a log2-based scale. ***P < 0.0001.
- Supplemental Figure S2
Effects of the reduction of AIB1 protein levels on endothelial cell phenotype. A: Flow cytometric analysis with annexin V–fluorescein isothiocyanate (FITC)/propidium iodide double staining of HUVECs 48 hours after infection with a control or AIB1/SRC-3 shRNA. The percentage of cells in late apoptosis is indicated. B: Cell proliferation measured by crystal violet assay. Values are given as the mean ± SEM of one of two independent experiments performed in triplicate. ***P < 0.0001 versus control. C and D: Repair of denuded endothelial monolayer areas was performed in the presence of 2 μg/mL mitomycin C. C: Representative images of denuded areas at 24 and 48 hours with the black line indicating the migration front of HUVECs. Scale bar = 0.1 mm. D: Quantitation of the closure of the denuded area. Values are given as the mean ± SEM of one of two independent experiments performed in triplicate. **P < 0.001 versus control.
- Supplemental Figure S3
Effects of AIB1/SRC-3 expression in fibroblasts in vitro. A: AIB1/SRC-3 overexpresssion in −/− murine embryonic fibroblasts.29Cells were infected with lentiviral vector ± FLAG-tagged AIB1. Top: Proliferation determined using Cell Titer Glo. Bottom: Western blot (WB) for AIB1 protein and actin. B: Depletion of AIB1 from NIH3T3 fibroblasts. Proliferation of NIH3T3 cells infected with a control shRNA or AIB1/SRC-3 shRNA #1 or #2. Values are given as the mean ± SEM of two independent experiments, performed in triplicate. C–F: Effects of depletion of AIB1/SRC-3 protein from Hs-27 cells. C: Western blot analysis for AIB1 and actin 48 hours after infection. The data are representative of two experiments. D: Proliferation of Hs-27 cells. Values are given as the mean ± SEM of one of two independent experiments performed in triplicate. E: Migration of Hs-27 cells after wounding of a confluent monolayer. Representative microphotographs at different times. The white line represents the migration front of the cells. F: Closure of the denuded area. Values are given as the mean ± SEM of one of two independent experiments performed in triplicate. ***P < 0.0001 versus control (analysis of variance).
- Supplemental Figure S4
CD31 and CD14 mRNA expression in Matrigel plugs. Quantitation of CD31 and CD14 mRNA by quantitative real-time PCR of mRNA prepared from Matrigel plugs 3 to 7 days after implantation. Values were normalized to CD31 or CD14 expression levels in the plugs on day 3. Values are given as the mean ± SEM (n = 3 animals per time point and genotype).
- Supplemental Figure S5
Histologic analysis of wounds. A: PCNA immunostaining of proliferating keratinocytes in the hyperproliferative epithelium (he) of skin wounds of AIB1/SRC-3+/+ and +/− mice on day 4 after wounding. Dotted lines depict the delimitation between the he and the granulation tissue (g). Scale bars: 0.2 mm. B: Quantitation of PCNA-positive nuclei. Values are given as the mean ± SEM (n = 3 animals per group). **P < 0.001 versus control. C: Immunohistochemical staining for AIB1/SRC-3 protein in mouse skin tissue sections and wound granulation tissue from +/+, +/−, and −/− mice. Scale bars: 0.2 mm (top); 0.1 mm (bottom). D: Representative H&E-stained sections of excisional skin wounds from AIB1/SRC-3+/+ and +/− mice at different times after wounding (4, 6, and 8 days). Magnified areas shown on the right are indicated. Scale bars: 0.2 mm. s, scab; e, epithelium; n, polymorphonuclear neutrophils; g, granulation tissue; asterisks denote fibrin clots; arrowheads point to capillaries.
- Supplemental Figure S6
Wound healing in transplanted skin from and to AIB/SRC-3+/+ and +/− mice. Full-thickness skin grafts from +/+ and +/− mice (donors) were grafted onto the backs of +/+ (A) and +/− (B) mice (recipients), respectively. Day 9 after grafting, a dermal biopsy punch (3-mm diameter) was used to generate full-thickness skin wounds through the center of the grafted skin in anesthetized animals (3 to 4 months old), and they were left to heal. Representative images of the grafts and wounds on day 3 are shown. Open wound areas were quantified daily for 5 days. Values are given as the mean ± SEM of the wound area (n = 3 animals per genotype). ***P < 0.0001 +/+ versus +/−.
- Supplemental Figure S7
AIB1 mRNA expression in skin wounds. Quantitative RT-PCR for AIB1 in skin wounds of AIB1/SRC-3+/+ or +/− mice 0 to 4 days after wounding. Values are given as the mean ± SEM (n = 3 animals per time point and genotype).
- Supplemental Figure S8
MMP activity in wounds. Quantitation of the fluorescence signal of MMP activatable fluorescence agent measured in wounds from AIB1/SRC-3+/+ and +/− mice 2 to 6 days after wounding. Values are given as the mean ± SEM (n = 4 animals per group).
- Supplemental Figure S9
Expression of angiogenesis-related genes in day 4 wounds of AIB1/SRC-3+/+ versus +/− mice. Confirmatory analysis of a gene expression survey in day 4 wounds using the mouse angiogenesis RT2 Profiler PCR array. Quantitative real-time PCR results relative to actin are shown. *P < 0.05 comparing wounds across genotypes.
- Supplemental Figure S10
Expression ratio of FGFR b and c splice isoforms in day 4 wounds of mice. mRNA expression of the b and c isoforms of FGFR1 to FGFR3 was analyzed by quantitative RT-PCR. The ratio of the b/c isoforms is shown. Values are given as the mean ± SEM (n = 3 animals per time point and genotype). *P < 0.05.
- Supplemental Figure S11
Effects of the reduction of AIB1 protein levels on FGF2-induced signaling of endothelial cells. HUVECs were transduced with shRNAs for either control or AIB1/SRC-3, and nontransduced cells were eliminated by 2 mg/mL puromycin treatment. After 4 hours of growth factor depletion in endothelial basal medium-2, cells were stimulated with or without 10 ng/mL FGF-2 for 10 minutes, and cell lysates were analyzed by Western blotting. Densitometry values were normalized to no FGF treatment for each shRNA treatment.
- Supplemental Table S1
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Funded by NIH/National Cancer Institute grants CA113477 (A.T.R.) and CA71508 (A.W.), by Department of Defense Breast Cancer Research Program grant BC083320 (C.D.C.), and by T32 National Cancer Institute training grant CA009686 (M.A.). Flow cytometry and time lapse microscopy were performed by Lombardi Comprehensive Cancer Center core facilities, supported in part by cancer center support grant CA051008 .
A.T.R. and A.W. contributed equally to this work.
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