Sc 6251

WA-25 (dihydroaustrasulfone alcohol) was synthesized by the Research Center of National Research Program for Biopharmaceuticals (Taipei, Taiwan) as described previously [23 (link)]. Quercetin and VEGF-A was obtained from Sigma Chemical (St. Louis, MO, USA). All drugs were dissolved in normal saline. Antibodies against VEGF (SC-152), VEGFR2 (SC-6251), and β-actin (SC-8432) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

EAhy926 cells were adjusted to a concentration of 1 × 10⁶ cells/ml. Cells were added in a six-well plate with 200 μl cells in each well. For Sunitinib treatment, cells were incubated in the presence of the indicated concentrations of Sunitinib in serum-free medium at 37 °C for 24 h. After washing the cells twice with PBS, cells were collected and incubated with mouse monoclonal anti-VEGFR2 antibody (sc-6251, Santa Cruz) on ice for 1 h. After washing and centrifugation, cells were resuspended and incubated with FITC-labeled goat anti-mouse secondary antibody (ab6785, Abcam) at room temperature for 1 h. After washing twice with PBS, fluorescent signals were collected with MACS Quant Data (Miltenyi Biotec, Germany). Three graphs were created for evaluating the data and creating a gate for the cells. The cells from the control without antibody were analyzed and events gated to remove debris from the analysis. This gate was used for all the samples. For the cell samples, the geometric means of values of events within the gate were calculated. The geometric mean of the control without antibody was subtracted from all of the sample geometric means to remove background noise.

HCT116 cells were seeded on a microcover slip and then transfected with pCMV6-Entry-NGB plasmids. After 48 h, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and then blocked with blocking buffer. Afterward, slides were incubated with primary antibody at 4 °C. After 20 h, the cells were incubated with Alexa Fluor 594- or 488-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (ab150113, ab150080, Abcam) for 1 h at room temperature in the dark. All slides were next counterstained with 4'-6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology). Photomicrographs were captured with a confocal laser scanning microscope. All assays were performed three times. Primary antibodies included anti-NGB (sc-133086, Santa Cruz Biotechnology), anti-GPR35 (TA313953, Origene technologies), anti-Myc tag (#2278; Cell Signaling Technology), anti-CD31 (#3528, Cell Signaling Technology), and anti-VEGFR2 (sc-6251, Santa Cruz Biotechnology).

Collected A549 and A549/GR cells were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet p-40, 50 mM Tris, pH 8.0, and a protease inhibitor cocktail). Thirty micrograms of each sample was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad, USA). Western blotting was performed with rabbit or mouse antibodies against Prx II: LF-PA0091 (AbFrontier, Seoul, South Korea), CD133: PA2049 (Boster Bio, CA, USA), Nanog: sc-293121, OCT3/4: sc-9081, Sox2: sc-365823 (Santa Cruz Biotechnology), VEGFR2: sc-6251, E-cad: sc-7870, Vimentin: sc-6260 (Santa Cruz Biotechnology), Shh: 2207 s, Gli-1: 3538 s, Notch 1: 3268 s (Cell Signaling Technology), CXCR4: YF-MA16239, STAT3: LF-MA30485,pSTAT3 (Tyr 705): LF-PA20474, pSTAT3 (Tyr-727): LF-PA20473 Hes-1: YF-MA11051, and β-Catenin: YF-MA10213 (AbFrontier). Protein expression levels were detected using Super signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, #34577).

Tissues underwent antigen retrieval by boiling in an electric pan with 10 mM citrate buffer, pH 6.0, for 40 min. Endogenous peroxidase blockade was done with 3% hydrogen peroxide in phosphate-buffered saline (PBS). Nonspecific binding sites were blocked with 10% goat serum diluted in PBS for 1 h in a humid dark room. The samples were then incubated overnight at 4°C for 12 h with the primary antibodies diluted in 1.8% bovine serum albumin (BSA) (VEGF: mouse, 1:150, ab1316, Abcam, USA; VEGFR-1: rabbit, 1:50, ab2350, Abcam, USA; VEGFR-2: mouse, 1:25, sc6251, Santa Cruz Biotechnology, USA). After removal of the primary antibody, the appropriate secondary antibody (VEGFR-1: biotinylated goat anti-rabbit-sc-2040 antibody, Santa Cruz Biotechnology; VEGF and VEGFR-2: biotinylated goat anti-mouse antibody-sc-2005, Santa Cruz Biotechnology) diluted 1:200 in BSA was added. For the negative control, the primary antibody was omitted. The samples were then incubated for 30 min in streptavidin-HRP (Biolegend #405210, USA) diluted 1:200 in PBS. Next, they were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA) diluted 1:100 in hydrogen peroxide for 20 min. Finally, the samples were counterstained with Harris hematoxylin, washed in running water, dehydrated in an alcohol series and xylene, and covered with coverslips mounted with Permount^® (Fisher Scientific).

Human KRAS-mutant NSCLC biopsy sections and linked clinical data (Supplemental Table 2) for 20 patients were obtained from the Zhengzhou University Cancer Biobank. Samples were dehydrated, formalin-fixed, and paraffin-embedded, and 5 μm serial sections were mounted onto glass slides. Sections were incubated with primary antibodies recognizing BACH1 (sc-271211, Santa Cruz Biotechnology, 1:200); VEGFA (sc-7269, Santa Cruz Biotechnology, 1:200); and VEGFR2 (sc-6251, Santa Cruz Biotechnology, 1:200) at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies (Zhong-shan Golden Bridge) for 1 hour. Next, the sections were stained with 3,3′-diaminobenzidine and hematoxylin. The stained sections were then scanned using a Panoramic Confocal microscope (3DHistech). Quantification of BACH1, VEGFA, and VEGFR2 staining intensity was performed using Aipathwell digital pathology AI-based image analysis software. Each sample was assigned a score on the basis of modified H-scores [H-scores =∑ (pi × i) = (percentage of weak intensity × 1) + (percentage of moderate intensity × 2) + (percentage of strong intensity × 3)] (33 (link)–36 (link)).

The expression levels of the CSps surface markers CD31 (1:200, GB11063-3, Servicebio), CD34 (1:200, ab81289, Abcam), CD90 (1:200, ab225, Abcam), CD105 (1:200, ab156756, Abcam), Sca-1 (1:200, ab51317, Abcam), and KDR (1:200, sc6251, Santa Cruz) were determined by flow cytometry. After a 3-day cultivation, cells and CSps from different substrates were obtained and digested into single cells. After incubation with the primary antibodies for 1 h and the corresponding secondary antibodies for 30 min, Alexa Fluor 488 goat anti-mouse IgG (1:200, ab150113, Abcam) or Alexa Fluor 488 goat anti-rabbit IgG (1:200, ab150077, Abcam) was used. The staining results were analyzed by flow cytometry (BD FACSCanto), and a negative isotypic control was used during the analysis.