61392-RG-2 (supplementary data) · cells were transduced with lentiviruses shRNA targeting all VCAN...
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61392-RG-2 (supplementary data) RhoGDI2 suppresses metastasis via reduction of versican expression and macrophage infiltration in murine models Neveen Said 1 , Steven C. Smith 1 , Marta Sanchez-Carbayo 2 and Dan Theodorescu 3,4 * 1 Department of Urology, University of Virginia, 2 Spanish National Cancer Institute (CNIO), 3 Departments of Surgery and Pharmacology, University of Colorado 4 University of Colorado Comprehensive Cancer Center. Type: Regular Article Key Words: bladder cancer, metastasis, RhoGDI2, versican, inflammation Running Title: RhoGDI2 Suppresses Bladder Cancer Metastasis * Corresponding Author: Dan Theodorescu, Departments of Surgery and Pharmacology and Comprehensive Cancer Center, University of Colorado, Aurora, Colorado 80045, USA. Phone: 303-724-7135; Fax: 303-724-3162; E-mail: [email protected] .
Transcript of 61392-RG-2 (supplementary data) · cells were transduced with lentiviruses shRNA targeting all VCAN...
61392-RG-2 (supplementary data)
RhoGDI2 suppresses metastasis via reduction of versican expression and macrophage infiltration in murine models
Neveen Said 1, Steven C. Smith 1, Marta Sanchez-Carbayo 2 and Dan Theodorescu 3,4 *
1 Department of Urology, University of Virginia, 2 Spanish National Cancer Institute (CNIO), 3 Departments of Surgery and Pharmacology, University of Colorado
4 University of Colorado Comprehensive Cancer Center.
Type: Regular Article
Key Words: bladder cancer, metastasis, RhoGDI2, versican, inflammation
Running Title: RhoGDI2 Suppresses Bladder Cancer Metastasis
* Corresponding Author: Dan Theodorescu, Departments of Surgery and Pharmacology and
Comprehensive Cancer Center, University of Colorado, Aurora, Colorado 80045, USA. Phone:
303-724-7135; Fax: 303-724-3162; E-mail: [email protected].
SUPPLEMENTARY FIGURES Supplement Figure 1. a. Engineered UMUC3 cells in Figure 3 were allowed to invade through 8-µm pore polycarbonate inserts coated with either matrigel, or confluent monolayers of primary human pulmonary microvascular endothelial cell (PMVECs) towards complete growth medium (CGM) or U937 in the bottom chamber. b. Monolayers of transfected/transduced cancer cells allowed to attract U937 cells through 3-µm pore inserts coated with either matrigel or confluent monolayers of PMVECs. Invading/attracted cells on the undersurface of the filters were counted. Bars represent mean ± SEM of three independent experiments performed in triplicates.*P<0.05, Students’t-test. Supplement Figure 2. RhoGDI2 and versican expression in T24/T24 cell lines. a. Western blots (WB) showing the expression of endogenous Rho-GDI2 protein in the two isogenic cell lines T24 and T24T. b. WB showing the expression of V1 and V3 versican in T24T and T24 cells were transduced with lentiviruses shRNA targeting all VCAN isoforms (shVCAN), an irrelevant target (non-target, NTsh) or c. retroviruses overexpressing V1, and V3 and their empty vector controls (VC), respectively. Equal protein loading was confirmed by tubulin (tub).
Supplement Figure 3. GDI2 modulates cancer cell-macrophage interactions through VCAN in T24/T24T isogenic cell lines. effect of depletion of versican in T24T (upper) and overexpression of V1 and V3 in T24 cells (lower) on their ability to: a. invade through 8-µm pore polycarbonate inserts coated with matrigel towards complete growth medium (CGM, lower left) or U937 lower right) in the bottom chamber. b. invade through 8-µm pore polycarbonate inserts coated with PMVECs towards complete growth medium (CGM, lower left) or U937 (lower right) in the bottom chamber. c. monolayers of T24T and T24 depleted of (upper) or overexpressing (lower) VCAN were allowed to attract U937 cells through 3-µm pore inserts coated with either matrigel or confluent monolayers of PMVECs. Invading/attracted cells on the undersurface of the filters were counted. Bars represent mean ± SEM of three independent experiments performed in triplicates.*P<0.05, Students’t-test.
Supplement Figure 4. Depletion of VCAN in T24T cells abrogates their metastatic capability. a. Photos of lung metastasis (circled in yellow) and scatter plot of the incidence and number of visible metastases (mets) 6W post-injection of T24T-NTsh and T24T-shVCAN cells developing after tail vein injection of 1x106 cancer cells in nude mice. *P<0.05, χ2 test; comparing the incidence, and **P<0.01, Student’s t test, comparing the number of mets. b. WB of the expression of VCAN isoforms in representative lung lysates (n=2) 6W post-injection of cancer cells c. Macrophages infiltration in metastatic foci (circled in yellow) and surrounding lung parenchyma was determined by mac2 immunostaining. Bars represent the mean±SEM of macrophage number, counted in 6 random high power fields (HPF); **P<0.01, Student’s t-test. d-e. Human IL-6, and CCL2 (hIL-6, hCCL2), murine IL-6, and CCL2 (mIL-6, mCCL2), and Cox2 activity were determined in lungs’ lysates. Bars represent mean±SEM, (n=3, performed in duplicates). *P<0.05, **P<0.01 Student’s t-test.
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