Xenograft Tumor Model in BALB/c Nude Mice

The A549 cells carrying the pcDNA3.1/luciferase vector (1 × 10⁶/100 μl PBS) were inoculated subcutaneously (s.c.) into 6-week-old female BALB/c nude mice (BALB/cAnN.Cg-Foxn1^nu/CrlNarl; n=11), and tumors were allowed to develop for 8 weeks. A randomized method was used to assign the mice into the experimental groups (control and MCT-1). The animal studies were conducted in accordance with the Animal Use Protocol approved by the National Health Research Institutes (NHRI-IACUC-104020-A). Tumor tissues were processed for immunohistochemistry analysis as previously described.^{27 (link)} Luciferin (150 mg/kg; PerkinElmer, Waltham, MA, USA) was intraperitoneally injected into mice to detect tumor progression using a Xenogen IVIS 200 bioluminescence imaging system (Caliper LifeSciences, Hopkinton, MA, USA).
The Abs for immunohistochemistry were diluted as follows: MCT-1, 1:500 (GeneTex, GTX117793); YY1, 1:400 (GeneTex, GTX62783); CD31, 1:200 (Abcam, ab28364); CD163, 1:100 (Abcam, ab189915); αSMA, 1:4000 (GeneTex, GTX112862); MnSOD, 1:2000 (Enzo Life Sciences, Inc., ADI-SOD-111-D); p53, 1:100 (Millipore, DAM1698716, Billerica, MA, USA); and phospho-EGFR, 1:100 (Cell Signaling Technology, #4407S).