Cxcr4

Expression of human NSCLC stem cell marker CD133 and CD44 was analyzed by flow cytometric study in A549 cells and spheroids by using anti-CD133-FITC and anti-CD44-APC antibodies (BD Biosciences). CD133⁺ and CD44⁺ CSCs were flow-cytometrically gated from spheroids on the basis of the cell surface phenotype. Mean fluorescence intensities of Oct-4-PerCP-Cy5.5, Nanog-PE, Sox-2- Alexa Fluor-647, Mrp1-FITC, Aldh1-FITC (BD Biosciences) were quantified^{22 (link)}. Mean fluorescence intensities of Akt, p-Akt, Cxcr-4, Mdr1, Integrin-α2, Integrin-α5, Integrin-β1, p-Fak (Santa Cruz Biotechnology, Inc.) were determined with respective primary antibodies conjugated with PE as previously described^{13 (link)}.

hBM-MSC seeded at a density 5 × 10³ cells on 24-well plates, stained with Molday ION Rhodamine B™ and CellTracker™ Green CMFDA, or transfected with mRNA eEGFP on 2nd and 7th day of culture were washed three times with PBS and fixed by 15-min incubation in 4% paraformaldehyde (PFA; MP BIOMEDICALS) in PBS and then again washed three times in PBS. Permeabilization was accomplished by 3-min incubation in 0.05% Triton X100 (Triton®X-100 Sigma Ultra, Sigma-Aldrich) in PBS. Non-specific binding was blocked using 3% of albumin from bovine serum (BSA; Sigma-Aldrich) for 90 min. The cells were washed with PBS, and they were incubated overnight at 4 °C with primary antibodies against CD90 (1:250, Sigma-Aldrich), CD44 (1:500, Santa Cruz), SSEA4 (1:100, Millipore), and CXCR4 (1:250, Santa Cruz). Then, cells were washed three times with PBS, and they were exposed for 90 min (RT in the dark) to goat anti-mouse IgG (H+L)-488 (Invitrogen) or -546 (Invitrogen) secondary antibodies. Additionally, cell nuclei were stained with 5 μM Hoechst 33258 (Sigma-Aldrich). The images were captured using fluorescent microscope Axiovert.A1 Zeiss (Carl Zeiss MicroImaging GmbH). Following acquisition, images were processed using the Zeiss Axiovert.A1 software package v. ZEN blue software (Carl Zeiss MicroImaging GmbH).

Cells were lysed in 1× Laemmli lysis buffer (62.5 mM Tris pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 0.02% bromophenol blue). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred by electroblot onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States). Each PVDF membrane was blocked with 5% non-fat dry milk or 5% BSA in Tris-buffered saline (TBS) Tween 20 (0.1%, v/v) buffer at room temperature for 1 h. Then, the membranes were incubated with the primary antibodies against HSP27(Enzo Life Sciences, United States), N-cadherin, β-catenin, Daxx, Vimentin, Akt, MMP2, p53, Mdm2 (Cell Signaling Technology, United States), β-actin, GAPDH, TERT, MMP1, CXCR4 (Santa Cruz, United States), CD44 (Invitrogen, United States), or GRP78 (Proteintech, United States) for 1 h to overnight depending on the antibodies. Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin (Ig) G was used as a secondary antibody. Immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, 32106).

Cellular phenotypic analysis was carried out by direct immunofluorescence with the following antibodies: LY6G (eBioscience, USA), F4/80 (eBioscience, USA), CD11b (eBioscience, USA), CXCR4 (Santa Cruz Biotech, USA), metallomatrix proteinase 9 (MMP9) (R&D Systems, USA), CD44 (Miltenyi Biotec, Germany), Sirpα (Biolegend, USA), CD47 (Miltenyi Biotec, Germany), CDH1 (Biolegend, USA), and CD55 (Miltenyi Biotec, Germany). All incubations were done in the presence of 50 µg/ml mouse IgG, the same isotype control antibody was always included as a negative control, and dead cells were excluded by 7-amino-actinomycin D staining (Sigma, USA). Flow cytometry was performed with a Gallios device (Beckman Colter, USA) and data were analyzed using Flowjo software (Tree Star, Inc., USA). Cells were counted using Flow-Count fluorospheres (Beckman Coulter, USA) following the manufacturer’s instructions.

For cytoimmunofluorescence (CIF), F_A7DOWN/P_A7UP, F_SHUS/P_NCEV, F/P cells and normal liver cells in vitro were incubated with SDF1 (Santa Cruz, CA, 1:75) and CXCR4 (Santa Cruz, CA, 1:75) polyclonal antibodies at 4 °C overnight followed by incubation with fluorescence secondary antibodies (DyLight 594 AffiniPure Donkey Anti-Rat/Mouse; Abbkine, USA, 1:50) at 37 °C for 90 min, and the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole diaminobenzidine (DAPI). Cytoimmunofluorescence was observed under laser confocal microscope, and Image J software was used to calculate average OD values.
Immunohistochemistry (IHC) was used to detect the expression of AnnexinA7, SDF1 and CXCR4 in F_A7DOWN/P_A7UP, F_SHUS/P_NCEV, F/P cells and normal liver tissue in vivo. A mouse monoclonal antibody against AnnexinA7 and rabbit polyclonal antibodies against SDF1 and CXCR4 were used at dilutions of 1:1500, 1:400 and 1:400, respectively. A Ready-to-use Elivision TM plus Polymer HRP (Mouse/Rabbit) IHC Kit (Fuzhou Mai Xin Biotech, China) was used as the secondary antibody. Protein expression levels were quantified based on the intensity and uniformity of nuclear/cytoplasmic staining, and ImageJ software was used to calculate average OD values.