Wzb117

Mice were randomly divided into two groups (n = 15), including an experiment group and a vehicle group. Both groups received orthodontic force application. The experiment group was injected with a specific GLUT1 inhibitor, WZB117 (S7927, Selleck).^{23 (link)} For systemic administration of WZB117, each mouse was injected intraperitoneally with WZB117 solution at 10 mg·kg⁻¹. The WZB117 solution was prepared by dissolving in dimethylsulfoxide (DMSO) (40 mg·mL⁻¹ in stock) and diluting in PBS to form 200 μL solutions for each injection. At the same time, all mice in the vehicle group received an injection of an equal volume of DMSO diluted in PBS. Mice were first injected one day before the application of mechanical force. Then, injections were performed every other day until the last day of force application.

Briefly, stable transfection cells (1.0 × 10⁷) were subcutaneously implanted into the bilateral flanks of 4-week-old nude mice. In the drug delivery experiments, ten days after subcutaneous injection and palpable tumors were present in all animals, the mice were then randomly divided into 3 groups, and 3-BrPA (Selleck) or WZB117 (Selleck) were then injected intraperitoneally every 2 days at the concentration of 5 mg/kg/d, and PBS as a control. The tumor growth was monitored and tumor volumes (mm³) were estimated as follows: tumor volume = L × W²/2, where L is the length and W is the width. Tumors were harvested when reached a diameter about 2.0 cm. Tumors were excised and rapidly put in formalin for further IHC analysis.

Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), and apatinib were purchased from MCE (NJ 08852, United States); WZB117 and S3I-201 were purchased from Selleckchem (Houston, TX, United States); Annexin V-FITC/PI apoptosis detection kit was purchased from KeyGen Biotech (Nanjing, China); recombinant human IL-6 was obtained from PeproTech (Rocky Hill, United States). Fetal bovine serum (FBS) and Dulbecco’s modified eagle medium (DMEM) were purchased from Gibco (Brooklyn, NY). All the other materials used were also of analytic reagent grade and were used without further purification. Antibodies against STAT3, hexokinase 2 (HK2), pyruvate kinase isozyme 2 (PKM2), PFKFB3, and hypoxia-inducible factor-1α (HIF-1α), goat anti-rabbit and anti-mouse immunoglobulin G (IgG) secondary antibodies, as well as goat anti-rabbit IgG H&L (Alexa Fluor 488), were obtained from Zen bioscience (Chengdu, China). Antibodies against phosphorylated (p)-STAT3 were purchased from Cell Signaling Technology (Danvers, MA, United States). The p-VEGFR2 antibody was from Affinity Biosciences (OH, United States).