Gp130

Standard Western blot analysis was performed on 10 μg of protein from the cell lines of interest using protocols as previously described [13 (link)]. PDGFRβ was purchased from Millipore (catalog # 06-495), PDGFRα (product # 9360) and GP130 from Cell Signaling Technology (product #3732), and α-IL6R from Thermo scientific (catalog #PA5-27975).

Human lung cancer cells, LNM35 and A549, were maintained in RPMI 1640 (Hyclone, Cramlington, UK), human breast cancer cells, MDA-MB-231 and T47D; human colon cancer cells HT-29, HCT-116, and HCT8/S11; and mouse gastric stem cells (MGSC) were maintained in DMEM (Hyclone, Cramlington, UK). All media were supplemented with antibiotics (penicillin, 50 U/ml; streptomycin, 50 µg/ml) (Hyclone, Cramlington, UK) and with 10% foetal bovine serum (Hyclone, Cramlington, UK). In all experiments, cell viability was higher than 99% using trypan blue dye exclusion. The culture medium was changed every 3 days, and cells were passaged once a week when the culture reached 95% confluence. PTC-209 was purchased from Xcess Biosciences Inc (Xcess Biosciences Inc, San Diego, CA). Frondoside A, camptothecin, cisplatin, oxaliplatin, and 5-fluorouracil were purchased from Sigma-Aldrich (Saint Louis, MO). Antibodies to β-tubulin, gp130, STAT3, phospho-STAT3 were obtained from Cell Signaling Technology (Cell Signaling, Beverly, MA). Bmi-1 antibody was obtained from Abcam (Cambridge, UK). Antibody to β-actin was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).

Cells were seeded in 24-well plates (3000 cells/well) overnight. For phosphorylated (p)-STAT3 staining, cells were treated with IL-6 (25 ng/ml) for 24 h (37 °C). Then, 4% paraformaldehyde was used to fix the cells for 20 min at room temperature, followed by incubation with 50 µl of normal goat serum (cat. no. AR0009, BOSTER Biological Technology Co., Ltd.) for 30 min at room temperature. The primary antibodies p-STAT3^Y705 (1:100; cat. no. 9145, Cell Signaling Technology, Inc.), gp130 (1:100; cat. no. MAB4681-SP, Novus Biologicals, LLC) and ART1 (1:100; cat. no. ab185293, Abcam) were added for incubation at 4 °C overnight, after which the fluorescent secondary antibody (red light, DyLight 549, 1:100, cat. no. A23340, Abbkine Scientific Co., Ltd.; and green light, DyLight 488, 1:100, cat. no. A23220, Abbkine Scientific Co., Ltd.) was added for incubation at room temperature for 1 h. DAPI staining was then conducted for 5 min. Images were captured using an AMG EVOS FL microscope. The average optical density of p-STAT3 and gp130, together with the colocalization rate of gp130 and ART1, were determined from three randomly selected images using ImageJ software (version 1.53a, National Institutes of Health). The Pearson correlation and overlapping coefficient results from ≥ 30 cells from three independent experiments are shown as a bar graph (error bars, SEM).

Human rhabdomyosarcoma cell lines (RH30, RD, or RH28) with persistent phosphorylated STAT3 were harvested after treatment with Bazedoxifene or DMSO at 60–80% confluence overnight, then lysed in cold RIPA lysis buffer containing protease inhibitors cocktail and phosphatase inhibitor cocktail. The lysates were subjected to 10% or 12% SDS-PAGE gel and transferred to a PVDF membrane. Membranes were incubated with a 1:1000 dilution of specific primary antibody and 1:10,000 HRP conjugated secondary antibody. Primary antibodies against phosphorylated STAT3 (Tyr705, p-STAT3Y705), STAT3, cleaved caspase-3, phospho-specific ERK1/2 (Threonine 202/Tyrosine 204), P-AKT, GP130, GAPDH and secondary antibody are all from Cell Signaling Technology (Beverly, MA, USA). IL-6R antibody was purchased from Abgent (San Diego, CA, USA), and JAK1 and ER-β antibody were purchased from R&D Systems (Gymea, NSW, Australia). Membranes were analyzed using enhanced chemiluminescence plus reagents and scanned with the Storm Scanner (Amersham Pharmacia Biotech Inc, Piscataway, NJ).

Bel-7404 and HepG2 cells were plated in 60-mm plated and grown to 70%
confluence. Cells were treated as indicated, washed with cold PBS, and lysed
using standard procedures. Cell lysates were resolved by SDS/PAGE and
transferred on to a PVDF membrane. The membrane was blocked with 5% skim
milk (m/v) in Tris-buffered saline with Tween-20 (TBST; 50 mM
Tris/HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20) at room temperature
for 1 h and incubated with IL-6R, gp130, STAT3, p-STAT3, or tubulin antibodies
(Cell Signaling Technology, Beverly, MA, U.S.A.) at 4°C overnight. The
membrane was then washed and incubated with horseradish peroxidase
(HRP)–conjugated secondary antibodies (Cell Signaling Technology)
following exposure to Immobilon™ Western Chemiluminescent HRP Substrate
(Millipore, New Orleans, LA, U.S.A.).