Journal: bioRxiv

Article Title: Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells

doi: 10.1101/791426

Figure Lengend Snippet: (A) Relative tumor volume of UZLX-GIST1 xenografts over the course of a 21-day treatment with vehicle, delanzomib or imatinib. Measurements represent the average of at least four tumors per group. (B) Histopathologic response of imatinib-sensitive UZLX-GIST1 xenografts to treatment with delanzomib in comparison with placebo-treated and imatinib-treated controls. Hematoxylin and eosin (H&E) as well as immunohistochemical staining for Ki-67, phosphorylated histone H3 S10 or cleaved PARP (10X magnification). Histopathologic response grade was determined according to Agaram et al. . Data shown in graph represent at least six tumors per group. Quantification of proliferation index (% Ki-67 positive cells), mitotic cells (pH3-positive cells per 10 high-power fields, HPF) and percentage of cleaved PARP-positive cells (brown staining, respectively) represents the average of at least six tumors per group. It is of note, that only intact, individual cells were included in the counts. Hence, the central debris area in the cleaved PARP stain (although massively positive) is not reflected in the accompanying graph. Columns, mean + SE; *, p≤0.05 in comparison to control; **, p≤0.01 in comparison to control; ***, p≤0.001 in comparison to control (Student’s t-test, 2-tailed). (C) Immunoblot analysis of UZLX-GIST1 xenografts treated with delanzomib (24 h) in comparison with placebo and imatinib-treated positive control. Each lane represents one xenograft (C, vehicle control; DLZ, delanzomib; IM, imatinib). Grouped immunoblot images are either cropped from different parts of the same gel or from a separate gel run with another aliquot of the same protein lysate. (D) Histopathologic response of imatinib-resistant GIST430 xenografts to treatment with delanzomib in comparison with placebo. (E) Immunohistochemical staining for phosphorylated histone H3 S10 in GIST430 xenografts treated with delanzomib or placebo control.

Article Snippet: Primary antibodies used for immunoblotting, immunofluorescence and immunohistochemistry were actin (Sigma), cleaved caspase 3 (Cell Signaling), CREB binding protein (CBP; Santa Cruz), cyclin A (Novocastra), pH2AX S139 (Millipore), H2AX (Bethyl), phospho-histone H3 S10, phospho-KIT Y719 (both Cell Signaling), Ki-67 (Thermo Scientific Neomarkers), KIT (Dako/Agilent), p27 Kip1 (Fisher/BD Biosciences Pharmingen), PARP (Invitrogen), cleaved PARP (Abcam) and mono-ubiquitin (BD Pharmingen).

Techniques: Comparison, Immunohistochemical staining, Staining, Control, Western Blot, Positive Control