Sc393099

Cellular extracts were prepared as previously described [5 (link)]. Protein concentrations in supernatants were determined using CD Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). We performed Ponceau S staining to confirm correct protein transfer. Western blots were performed with 20–30 μg of total protein extract, using antibodies against: E2F1 (1:400, sc-256 Santa Cruz, CA, USA), E2F2 (1:400, sc-633 Santa Cruz), TK1 (1:1000, A5-29686 Invitrogen), DCK (1:400, sc-393099 Santa Cruz), TS (1:400, sc-3930945 Santa Cruz), P-CHK1 Ser345 (1:1000, 2348 Cell Signaling, Danvers, MA, USA), CHK1 (1:400, sc-7898 Santa Cruz), P-RPA Ser4/8 (1:1000, A300-245A Bethyl, Waltham, MA, USA), RPA (1:1000, ab2175 Abcam, Cambridge, UK), Phospho-CDK1 Tyr15 (1:1000, 9111 Cell Signaling), CDK1 (1:1000, ab18 Abcam), HSP90 (1:2000, sc-13119 Santa Cruz). Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse (1:4000, sc-3697 Santa Cruz) or anti-rabbit (1:4000, sc-2030 Santa Cruz) IgG antibodies, followed by chemiluminescence detection (ECL, Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with a ChemiDoc camera (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry-based quantification was performed using Fiji software. Relative optical density was calculated by dividing the densitometry of the protein of interest with the respective loading control.

Immunohistochemical staining and analysis was performed as previously published^{[30 (link)-32 (link)]}. Briefly, Ki67 proliferation indices were determined by counting the number of Ki67 positive tumor cells in 10 individual 40x fields and dividing that number by the total number of tumor cells. The data are expressed as the mean ± S.E.M. of two independent experiments. Expression indices of RRM1, RRM2, hENT1, hCNT1, CDA, dCK, ALDH1, CD133, and CXCR4 were calculated by assigning a staining intensity of 0, 1, 2, or 3 to each specimen, and multiplying this intensity by the percent of tumor cells expressing the protein of interest^{[33 (link)]}. Overall scores ranged from 0 to 300. Data were derived from photomicrographs taken with Olympus BH-2 microscope with DP71 camera and DPS-BSW v3.1 software. Primary antibodies were obtained from multiple sources: Ki67 (ab92742, abcam, Cambridge, MA, USA), RRM1 (NBP2-49415, Novus Biologicals, Littleton, CO, USA), RRM2 (ab57653, abcam), hENT1 (SAB5500117, Sigma-Aldrich, St. Louis, MO, USA), hCNT1 (NBP2-30857, Novus Biologicals), CDA (ab82346, abcam), dCK (sc393099, Santa Cruz, Dallas, TX, USA), ALDH1 (sc166362, Santa Cruz), CD133 (CS#86781T, Cell Signaling, Danvers, MA, USA), CXCR4 (TA305935, Origene, Rockville, MD, USA).