PMC

A. U2OS_EV or U2OS_UXT cells were treated with or without 2-DG at the indicated concentration for 48 h. Cell viability was determined and the data are means ± SD from 3 experiments performed in triplicates. B. U2OS_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM) for 6 h followed by irradiation at the indicated dose. Cell numbers were determined 72 h after irradiation. Data are means ± SD from 3 experiments performed in triplicates. C. U2OS_EV or U2OS_UXT cells were pre-treated with vehicle (Veh) or 2-DG (2mM) for 6 h followed by irradiation at a dose of 4Gy. The cells were fixed 1 h after irradiation and stained with γH2AX. The numbers of γH2AX-positive cells were counted from 5 random fields. Data are means ± SD from 3 experiments performed in triplicates.

A. U2OS_UXT cells were subjected to Cignal Finder™ 10-Pathway Reporter Arrays (Qiagen). The results are from 3 independent experiments performed in triplicates. The numbers are means ± SD. B. U2OS_EV or U2OS_UXT cells were analyzed by immunostaining with an anti-p65 (Cell Signaling, MA) co-stained with DAPI. Representative images are shown. C. Cell lysates prepared from U2OS_EV or U2OS_UXT cells were analyzed by Immunoblot with the indicated antibodies. D. U2OS_EV or U2OS_UXT cells that were transfected with either siRNACtrl or siRNAp53 were treated with or without Nutlin-3a (10 μM 3 h). The cells were harvested and subjected Western analysis using the indicated antibodies.

A. A result of a yeast 2-hybrid assay indicating a positive clone pull out by a bait of MDMX but not MDM2. B. 293 cells were coexpressed with plasmids encoding a Flag-UXT and MDMX. The cells were harvest 24 h after transfection and subjected to reciprocal immunoprecipitations with an anti-Flag (M2 Sigma) or MDMX (Bethyl lab), with lgG included as a control. The immunoprecipitates were analyzed by Western analysis using anti-Flag or anti-MDMX antibodies. C. U2OS cells were transfected with either GFP-UXT or RFP-MDM alone to together. The cells were fixed 24 h later. Images were taken under a fluorescent microscope and representative images including overlay ones are shown. D. U2OS cells were subjected to anti-MDMX immunoprecipitation with LgG included as a control. The immunoprecipitates were immunoblotted with either anti-MDMX or anti-UXT. E. 293 cells were transfected with a control vector or 1 μg MDMX with 1 or 3 μg of Flag-UXT. The cells were harvested 24 h later and analyzed by Western blot with the indicated antibodies.

Cell culture media were collected from an equal number cells. The concentrations of lactate A. and glucose B. were determined by a colorimetric kit (BioVision). The numbers are means ± SD from 3 experiments performed in triplicates. C. U2OS_EV or U2OS_UXT cells were incubated with [1, 2-¹³C]-glucose for 15 min prior to metabolite extraction and targeted LC-MS/MS analysis. The ratio of ¹³C labeled to unlabeled (¹²C) metabolites was measured by LC-MS/MS are presented as mean ± SD over 3 independent samples. Metabolites with P values < 0.05 for pair-wise comparisons are shown. D. U2OS_EV or U2OS_UXT cells were harvested and mRNA isolated. Transcripts of the indicated genes were determined by quantitative RT-PCR. Data are means ± SD from 3 experiments performed in triplicates. E. U2OS_EV or U2OS_UXT cells were treated with or without Capsaicin (100 μM 3 h). qRT-PCR or Western determined the expression levels of HIF-1α transcripts and protein, respectively.

A. U2OS cells were transfected with either control siRNA (siRNACtrl) or 2 independent siRNA sequences targeting different region of the UXT gene. The cells were harvested 48 h later and analyzed for protein expression by Immunoblot using the indicated antibodies. B. UOS cells stably expressing an empty vector (EV) or UXT were established. The U2OS_UXT cells were treated with or without MG132 (10 μM) for 6 h prior to harvesting for Western analysis using the indicated antibodies. C. U2OS cells were transfected as in A and harvest 50 h later for Western analysis using the indicated antibodies. D. U2OS cells transfected with each of siRNA(1, 2 or 3) or 2 different patches of UXT overexpressing U2OS cells (UXT#1 or #2) were measured the rate of cell proliferation using a proliferation assay kit (Gibco). E. Balb C nude mice (4–6 weeks) were subcutaneously implanted with 2 × 10⁶ U2OS_EV or U2OS_UXT cells. The tumors were harvested 8 weeks later and the volumes were measured. The numbers are averages from a group of 5 mice ± standard deviation. F. The harvested tumor tissues were subjected to immunohistochemistry staining with an anti-Ki67 antibody. The representative images were shown.