Journal: Cancers

Article Title: Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors

doi: 10.3390/cancers15010259

Figure Lengend Snippet: Impact of CNVs in oncogenic-associated genes on corresponding protein levels. CNV-protein links were evaluated by Western blots on tumor lysates from passages (P1, P2, and P3) of the OS PDXs (HT72, HT77, HT87, and HT96). Baseline levels of selected proteins of interest were: RAD21, MYC (c-MYC), p53, Cyclin D3, Cyclin E1, p16 INK4A , PTEN, RAC1, HSP90Aβ1, γH2AX, total H2AX, p-RB1 (S795), total RB1, BRD4, CDK4, and CDK6. Vinculin served as the loading control and was probed from lysates extracted in urea or RIPA buffers since proteins of interest were efficiently extracted in different buffers (see materials and methods). Human vertebral mesenchymal stem cells (HVMSCs) and human osteoblasts-femoral (HOB-F) were included as normal healthy control cells. This experiment was conducted one time. Copy numbers for genes containing CNVs are the top of each immunoblot.

Article Snippet: The following antibodies were diluted in either 5% non-fat dry milk or 5% BSA in TBS-T per manufacturer’s instruction and used for detection: rabbit anti-RAD21 (130 kDa, cat# 4321, Cell Signaling Technology, Boston, MA, USA); mouse anti-c-MYC [9E10] (67 kDa, cat# sc-40, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); mouse anti-p53 [D0-1] (53 kDa, cat# sc-126, Santa Cruz Biotechnology Inc.); mouse anti-Cyclin D3 (31 kDa, cat# 2936, Cell Signaling Technology, Boston, MA, USA); mouse anti-Cyclin E1 (48 kDa, cat# 4129, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDKN2A/p16 INK4A (17kDa, cat# ab108349, Abcam, Waltham, MA, USA); rabbit anti-PTEN (54 kDa, cat# 9559, Cell Signaling Technology, Boston, MA, USA); rabbit anti-RAC1 (21 kDa, cat# 4651, Cell Signaling Technology, Boston, MA, USA); rabbit anti-HSP90Aβ1 (96 kDa, cat# NBP2-68937, Novus Biologicals, Centennial, CO, USA); anti-phospho-H2AX serine 139 [γH2AX-Ser139] (15 kDa, cat# 2577, Cell Signaling Technology, Boston, MA, USA); rabbit anti-total H2A.X [D17A3] (15 kDa, cat# 7631, Cell Signaling Technology, Boston, MA, USA); rabbit anti-RB1 [D20] (110 kDa, cat# 9313, Cell Signaling Technology, Boston, MA, USA); rabbit anti-phospho-RB1 Ser795 (110 kDa, cat# 9301, Cell Signaling Technology, Boston, MA, USA); rabbit anti-BRD4 [E2A7X] (200 kDa, cat# 13440, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDK4 [D9G3E] (30 kDa, cat# 12790, Cell Signaling Technology, Boston, MA, USA); rabbit anti-CDK6 [D4S8S] (36 kDa, cat# 13331, Cell Signaling Technology, Boston, MA, USA); rabbit anti-vinculin [E1E9V] (124 kDa, cat# 13901, Cell Signaling Technology, Boston, MA, USA), and rabbit anti-GAPDH [14C10] (37 kDa, cat# 2118, Cell Signaling Technology, Boston, MA, USA).

Techniques: Western Blot, Control