Ab57653

The 211 tissue samples were formalin-fixed and paraffin-embedded. The immunohistochemistry was conducted using an Envision Detection System (DAKO, Denmark) according to the manufacturer's instructions as described previously (13 (link)). We used the following commercial antibodies against RRM1 (10526-1-AP, Proteintech, 1:500), RRM2 (ab57653, Abcam, 1:200), and RRM2B (ab8105, Abcam, 1:500) for immunohistochemistry. PBS was used as a negative control.
To determine the score of each slide, at least eight individual fields at 200× were selected, and 100 cancer cells were counted in each field. Cells with cytoplasmic and/or nuclear immunoreactivity of RRM1, RRM2, and RRM2B were considered positive. The immunostaining intensity was divided into five grades: 0, negative; 1, weak; 2, moderate; 3, strong; and 4, very strong. The proportion of positive-staining cells was also divided into five grades: 0, <5; 1, 6–25; 2, 26–50; 3, 51–75; and 4, >75%. The IHC scores were generated by multiplying the intensity score and the proportion score. To avoid observer bias, and for consistency, the value of immunostaining intensity and the percentage of positive-staining cells for all the slides were evaluated independently by two different well-trained observers blinded to the clinical data.

The protein concentration was detected by bicinchoninic acid assay. After mixing with loading buffer, the proteins were denatured by boiling. Next, the proteins were separated by electrophoresis and transferred into polyvinylidene difluoride membranes. Subsequently, the membranes were incubated with the primary antibodies and then the secondary antibody goat anti-mouse anti-IgG H&L (HRP) (1/2000, ab205719, Abcam). Then, the membranes were developed using the enhanced chemiluminescence. The levels of Ki67 (1/1000, ab16667, Abcam, Cambridge, MA, USA), Cleaved Caspase-3 (20 µg/mL, ab32042, Abcam), Bcl-2 (1/500, ab692, Abcam), RRM2 (1 µg/mL, ab57653, Abcam, Cambridge, MA, USA), AKT (1/500, ab8805, Abcam), phosphorylated AKT (p-AKT, 1/500, ab38449, Abcam), phosphatidylinositol 3-kinase (Pl3K) (1/500, ab154598, Abcam), p-Pl3K (1/500, ab182651, Abcam) were determined, with GAPDH (1/500, ab8245, Abcam) acting as the internal reference. The quantification of band intensity was processed using Image-Pro Plus 6.0 software (Olympus, Tokyo, Japan).

FFPE tissue blocks from 12 patients who had undergone resection were used for immunohistochemical staining (IHC) staining. Paired tumor and adjacent samples were used for staining of EGLN1 (RE6068, HUABIO, Hangzhou, China), MIF (ab65869, Abcam, Cambridge, MA, USA), PANX1 (12595-1-AP, Proteintech, Wuhan, China), and RRM2 (ab57653, Abcam). The primary antibody of EGLN1 was diluted at 1:100, others were diluted at 1:200 and incubated at room temperature, and then the secondary antibodies were added for incubation. All the staining processes were carried out on the IHC System (Roche, Basel, Switzerland) following the manufacturer’s instruction.

Whole-cell protein extracts were prepared using RIPA lysis buffer (50  mM Tris, pH  7.4; 150 mM NaCl; 1 mM each of NaF, NaVO4 and EGTA; 1% NP40; 0.25% sodium deoxycholate; 0.2 mM phenylmethylsulphonyl fluoride; 1 μg/ml each of antipain, aprotinin and chymostatin; 0.1 μg/ml leupeptin; 4.0  μg/ml pepstatin) and detected by Western blot analysis as described [16 (link)]. Antibodies used were mouse monoclonal to RRM2 (ab57653, Abcam, Cambridge, U.K.), mouse monoclonal to caspase-3 (ab13585, Abcam, Cambridge, U.K.), mouse monoclonal to β-Actin (Sigma-Aldrich, St. Louis, MO, U.S.A.) and HRP-conjugated secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).

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).