Vimentin

Cells were grown on glass cover lips in the presence or absence of flubendazole for 24 hr. After fixed in 4% (v/v) formaldehyde/PBS, Cells were permeabilized (0.5% TritonX-100/PBS) and blocked for 1 hr in 5% BSA/PBS. IF was performed at room temperature using Vimentin (Merck Millipore, 1:200), Keratin 18 (Cell Signaling Technology, 1:100), α-tubulin (Santa Cruz, 1:400) and γ-tubulin (Sigma Aldrich, 1:800) antibodies. Alexa-conjugated anti-IgG antibodies (Invitrogen Corp, 1:200) were used for secondary detection. DAPI (Sigma Aldrich, 1 μg/ml) was used for nuclear staining. Images were acquired using an Olympus microscope.

Cells were washed twice with PBS and lysed in RIPA buffer (Beyotime, Haimen, China) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime). Protein lysates were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and incubated with primary antibodies for 24 h at 4°C. Membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, and immunoreactive bands were visualized with ECL chromogenic substrate (ComWin Biotech Co., Beijing, China). GAPDH was used as an internal control. Primary antibodies were as follows: E-cadherin (Ab133597; Abcam, Cambridge, UK), vimentin (CBL202; EMD Millipore, Billerica, MA, USA), β-catenin (Ab32572; Abcam), c-myc (Ab32072; Abcam), and GAPDH (ENM0215; Elabscience, Bethesda, MD, USA).

To validate HIF-1α nuclear localization, cells were incubated with 100 μM CoCl₂ for 24 h to induce hypoxia. Immunocytostaining was performed as previously described [41 (link)]. The following primary antibodies were used: HIF-1α (1:500; GeneTex, Irvine, CA, USA) and vimentin (1:10,000, Merck KGaA, Darmstadt, Germany). The following secondary antibodies were used: Alexa 568-conjugated goat anti-mouse IgG antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (both diluted 1:1000; Life Technologies). All images were acquired using the same settings on the confocal microscope (Carl Zeiss) and analyzed using ZEN 3.0 software (blue edition; Carl Zeiss). Samples incubated without a primary antibody were used as a negative control.

Cells were fixed in PBS containing 4% paraformaldehyde/4% sucrose for 15 min. Cells were permeabilized for 5 min at room temperature in 0.25% Triton-X-100 in PBS, washed twice with PBS, and incubated for 30 min at 37 °C in PBS containing 10% BSA. Cells were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 3% BSA. Antibodies used were as follows: mouse anti alpha-SMA (1:200, Invitrogen, Carlsbad, CA, USA); Myosin-IIB (1:1000, BioLegend, San Diego, CA, USA); YAP1 (1:50, Proteintech, Manchester, UK); TOM20 (1:80, Santa Cruz Biotech., Dallas, TX, USA); MCL-1 (1:800, Cell Signaling Technology, Danvers, MA, USA); Vimentin (1:100, Merck, Darmstadt, Germany). Actin labeling were performed with Alexa 488-conjugated Phalloidin (1:100, Cell Signaling Technology, Danvers, MA, USA). After washing, cells were incubated for 90 min at room temperature with the appropriate Alexa 488-conjugated secondary antibodies diluted in PBS containing 3% BSA. Cells were washed with PBS and mounted with ProLong Diamond Antifade Reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Fluorescence images were acquired with Nikon A1 Rsi Inverted Confocal Microscope (Nikon, Tokyo, Japan) with NIS-Elements software (Nikon). Images set position were generated randomly through NIS-Elements JOBS module (Nikon).

The cells were washed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Sigma-Aldrich, St. Lois, MO, USA). Western blot analyses were conducted as previously described [15 (link)]. Antibodies to the following proteins were used: ZEB1 and ZEB2 (Novusbio, Littleton, CO, USA), cyclin-D1 (Merck, Darmstadt, Germany), N-Cadherin (Merck), E-cadherin (Merck), vimentin (Merck), CD200 (R&D systems, Minneapolis, MN, USA), CD200R1 (R&D systems), c-Myc (Cell Signaling Technology, Danvers, MA, USA), β-catenin (Merck), Fibronectin (Merck), and β-actin (Santa Cruz, Dallas, TX, USA). For flow cytometry analysis, all cells (1 × 10⁷) or dissociated tumors were incubated for 15 min in the dark with anti-mouse CD16/CD32 antibody (BD Biosciences, San Diego, CA, USA). Cells (1 × 10⁷) were incubated for 30 min with anti-human CD200 PE-Cy7-conjugated antibody (BD Biosciences) or anti-mouse CD200 PE-conjugated antibody (BioLegend, San Diego, CA, USA). After washing again with FACS buffer, the MEER cell lines were resuspended in FACS buffer and analyzed. Densitometry readings/intensity ratio of each band was performed by using ImageJ software (ImageJ, NIH, Bethesda, MD, USA). Details information of western blot could be found in Supplementary Figure S7.

After incubated with varying concentration of flubendazole and vehicle (DMSO) for 48 hr, cells were harvested and lysed in RIPA buffer. Protein concentration was determined by Bradford assay. Briefly, cell lysates (20 μg) were separated by SDS-PAGE, transferred onto nitrocellulose membrane (Merck Millipore). The membranes were blocked and exposed to cyclinB1 (Becton Dickinson, 1:2000), cyclinE (Becton Dickinson, 1:1000), p-cdc2 (Tyr15, Cell Signaling Technology, 1:2000), p-cdc2 (Thr161, Cell Signaling Technology, 1:2000), cdc2 (Cell Signaling Technology, 1:1000), c-MYC (Cell Signaling Technology, 1:1000), OCT4 (Santa Cruz, 1:200), SOX2 (Shanghai Sangon Company, 1:500), NANOG (Abcam, 1:2000), Keratin 18 (Cell Signaling Technology, 1:500), cyclinD1 (Becton Dickinson, 1:500), N-cadherin (Abcam, 1:1000), Vimentin (Merck Millipore, 1:2000) and β-catenin (Merck Millipore, 1:2000) antibodies, followed by incubation with appropriate secondary antibodies (Thermo Fisher Scientific, 1:5000). Proteins were visualized with the ECL system from Bio-Rad. The Western blots shown were representative of at least three independent experiments. GAPDH (Kang Chen bio-tech, 1:5000) was used as the loading control.