Stat3

The biological activity of recombinant human interferon (IFNcon1, InterMune) was expressed in terms of international reference units/ml using the human NIH reference standard [47 (link)]. Antibodies against the following proteins were used: STAT1, STAT2, PTEN, IVNS1ABP, PI3KCA, EGFR, c-Myc, NMI, PD-L1 and actin (Santa Cruz Biotechnology); STAT3 (BD Transduction Laboratories); PDCD4, (Abcam) and FBXO11 (Novus Biologicals). MT330 (UTHSC Department of Neurosurgery), and SJG2 (St. Jude Children's Research Hospital) cell lines were grown in DMEM containing 10% Fetal Bovine serum (Atlanta Biologics) supplemented with penicillin (100 IU/ml) and streptomycin (100 mg/ml) at 37 °C with 5% CO₂.

Antibodies to c-Myc (sc-7274), cyclin D1 (sc-753), ErbB-3 (sc-285), Lyn A/B (sc-764), Fyn (sc-16), Src2 (sc-18), VEGF-A (sc-53462), E2A.E12 (sc-349), and ZEB1(sc-10572) (Santa Cruz Biotechnology), p27^Kip1 (BD-Pharmingen 554069), ALDH1 (BD, 661194), Stat3 (BD-Transduction Laboratories, S21320), Twist1/2 (Gene Tex, GTX127310), pY705-Stat3 (Cell Signaling Technology, #9131), Snail-1 (Cell Signaling Technology, LF062), CK5 (ABCAM, ab52635), pY418-Src (Invitrogen, 44660G), GAPDH (MAB374) and MMP9 (AB19016) (Millipore), PARP (Biomol, SA-249 clone C-2_10), β-actin (A5441), TAZ (HPA007415), and hydrocortisone were from Sigma-Aldrich, and CD44 (clone HP 2/9) was a gitf from Dr. F. Sanchez-Madrid (University Hospital La Princesa, UAM) [17 (link)], Matrigel^TM (Corning). Secondary horseradish peroxidase-conjugated antibodies, and B27 (Life Technologies). EGF, and bFGF (PeproTech EC Ltd). Fetal Calf Serum (FCS), Acrylamide/Bisacrylamide, SDS and ammonium persulfate (Bio-Rad Laboratories). ECL (GE Healthcare Biosciences). BCA protein assay (Thermo Scientific).

SNU-16 parental and resistant cells were seeded in six-well plates at a density of 0.5 × 10⁶ cells per milliliter in an inhibitor-free medium. After 24 h, cells were lysed and the level of proteins was examined by Western blotting according to the protocols provided by the antibody suppliers. The antibodies against MET, EGFR, HER2, extracellular-signal-regulated kinase (ERK), phosphorylated AKT, phosphorylated EGFR, phosphorylated ERK (pERK), phosphorylated FGFR, phosphorylated FGFR substrate, and phosphorylated signal transducer and activator of transcription (STAT3) were purchased from Cell Signaling Technology, β-tubulin and AKT were from Millipore, E-cadherin, STAT3, and β-catenin were from BD Biosciences, FGFR2 was from Abnova, and vimentin was from Calbiochem. All experiments were performed at least twice.

Oxaliplatin was purchased from Sanofi Pharmaceutical Company (NY, USA). The procedure for synthesizing the CTSS inhibitor RJW-58 was described previously 14 (link). The following primary antibodies were purchased: Cathepsin S (Thermo Fisher catalog: #PA5-81369), EBF (Santa Cruz, #sc-137065), ATF-3 (Biossua, #BS-0519R), α-tubulin (Sigma, MO, USA), IRF-1 (Bio-Rad, #VPA00801), CBP (GeneTex, #GTX101249), Iba1 (Abcam, #ab-5076), NeuN (Novus, #NBP2-10491), IL-10 (Abcam, #ab189392), STIM1 (Cell signaling (D88E10), #5668S), CD11b (Abcam, #ab52478), CD45 (BD Biosciences, #553079), STAT3 (BD, #50402), P-STAT3 (Cell signaling, #9145S), NFκB (Cell signaling, #4727S), OR5B3 (Abcam, #ab186624), OR5M3 (LSBio, #LS-C200406), and β-actin (GeneTex, #GTX109639). Horseradish-peroxidase-conjugated secondary antibodies were purchased from GeneTex (#GTX213110, #GTX213111, #GTX224125, and #GTX232040).

GICs were lysed at 4°C for 30 min in RIPA buffer (Sigma Aldrich, St Louis, MO) supplemented with protease inhibitor (Sigma Aldrich, St Louis, MO) and phosphatase inhibitor (BioTools, Jupiter, FL), followed by centrifugation (10,000xg for 10 min). Protein extracts (50μg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA), and immunoblotted with the following antibodies: STAT3 and pS727-STAT3 (BD Biosciences, San Jose, CA), pY705-STAT3 (Abcam, Cambridge, A), STAT1 and STAT5 (Cell Signaling, Danvers, MA), and Actin (Santa Cruz Biotechnology, Dallas, TX). Following addition of IRDye800CW goat anti-mouse IgG or IRDye680 goat anti-rabbit IgG, blots were visualized on an Odyssey infrared imaging system (LICOR Biosciences, Lincoln, NE).