Xav939

Recombinant human IL-17A was purchased from PeproTech (USA). Rat PE-conjugated
anti-mouse CD86 and rat FITC-conjugated anti-mouse CD206 were purchased from
BioLegend (USA). Rabbit anti-GSK-3β, Arg1, β-catenin, active-β-catenin (ABC),
phospho-STAT1 (signal transducers and activators of transcription 1), and
phospho-STAT3 antibodies were products of Cell Signaling Technology (USA).
Rabbit anti-STAT6 and phosphor-STAT6 antibodies were purchased from Affinity
Biosciences (USA). Rabbit anti-iNOS antibody was purchased from Abcam (USA),
rabbit anti-p21 was a product of Santa Cruz Biotech (USA). Rabbit anti-STAT3,
SOCS3, BCL-XL, c-Myc, TCF-4, β-actin, and mouse anti-Cyclin D1 antibodies were
purchased from Proteintech (China). The Wnt signaling inhibitor XAV939 was
purchased from Santa Cruz Biotech.

Leptin was purchased from ProSpec‐Tany TechnoGene (Rehovot, Israel). XAV939 (a specific inhibitor for β‐catenin signalling pathway) was purchased from Santa Cruz (Santa Cruz, CA, USA). Diphenyleneiodonium chloride (DPI, an inhibitor for NADPH oxidase signalling pathway) and thioacetamide (TAA) were from Sigma‐Aldrich (St. Louis, MO, USA). Ly294002 (a specific inhibitor for phosphoinositide 3‐Kinase, PI3K) and cyclopamine (a specific inhibitor for Hedgehog signalling pathway) were from selleck Chemicals (Houston, TX, USA).

Cells were plated into 96-well white-walled plates (Greiner Bio-One) at 1×10³ cells/well and allowed to adhere for 24 hours before drug treatments. The cells were then exposed to serial dilutions of doxorubicin (0–10 µM, LC Labs) and were placed in either normoxic or hypoxic conditions (as described above). After an additional 72 hours, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability assay (Promega). Data were normalized to an untreated control well and graphed, and half maximal inhibitory concentration (IC₅₀) values were calculated from the dose-response curve as the concentration of doxorubicin that produced a 50% decrease in the mean luminescence relative to untreated control wells. Statistical tests (two-tailed paired t-test and two-tailed two-sample t-test) were performed using R with significance set at α = 0.05. The tankyrase inhibitor XAV939 (Santa Cruz) and porcupine inhibitor IWP-2 (Sigma Aldrich) were used at a 10 µM concentration, with dimethyl sulfoxide (DMSO; Fischer) alone used as a control.