Additional file 2: of Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

Mice Null for the Deubiquitinase USP18 Spontaneously Develop Leiomyosarcoma. (DOCX 115 kb

Additional file 1: Table S1. of Mice null for the deubiquitinase USP18 spontaneously develop leiomyosarcomas

Antibodies used for immunohistochemistry studies. Table S2. Immunohistochemical staining of USP18 in clinical leiomyosarcoma samples. Figure S1. A: Dystrophic calcifications in USP18 null mice. A representative image of a USP18-/- mouse is shown. B: KHC-2 cells with reconstituted USP18 expression maintained expression for the duration of growth in mice. Immunoblot analysis of protein isolated from 4 control and 4 USP18 overexpressing independent orthotopic sarcomas harvested from mice. The immunoblot showed representative analysis of KHC-2 cells and this finding was also seen in KHC-1 cells (data not shown). Figure S2. USP18 null leiomyosarcoma cell lines are sensitive to treatment with the JAK2-STAT3 inhibitor, JSI-124. A: Immunoblot analysis of pSTAT3, STAT3, CDK4 and USP18 levels in KHC-1 cells with and without stably restored USP18 activity. B: Growth analysis of KHC-1 cells with JAK2-STAT3 inhibitor, JSI-124. Similar effects were seen in KHC-2 cells (data not shown). Validation of JSI-124 repression of JAK2-STAT3 pathway C: Immunoblot analyses of phosphorylated JAK2 (pJAK2), JAK2, and actin with relative level of pJAK2/JAK2 calculated relative to control. D: Immunoblot analysis of phosphorylated STAT3 (pSTAT3), STAT3, cyclin D1 and actin levels. Figure S3. USP18 null leiomyosarcoma cell line KHC-1 and human leiomyosarcoma cell line SK-LMS-1 growth in response to interferon-β (500Units/ml IFNB) or doxycycline (0.2ΟM Dox) treatment over 3 days. Results expressed as fold relative to vehicle treated cells. Each experiment was performed in triplicate 3 separate times. Figure S4. USP18 null leiomyosarcoma cell line KHC-1 with restored USP18 expression did not affect response to IFNb (500Units/ml). Results expressed as fold relative to vehicle treated cells. (N.S. = not significant). (PPTX 2131 kb