Chemicals and antibodies
Lipofectamine 2000 transfection reagent and TRIzol LS Reagent were purchased from Invitrogen (Grand Island, NY, USA). The DAB substrate kit for peroxidase was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Antibodies against Gli1, ERα, E-cadherin, Vimentin, Nanog, Bmi-1, SOX2, Smo and β-actin were from Cell Signaling Technology (Danvers, MA, USA). Anti-ALDH antibodies were from BD (Franklin Lakes, NJ, USA). Anti-Shh antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Amino-terminal Shh (ShhN) peptide was from R&D Systems (Minneapolis, MN, USA). Unless otherwise noted, all other chemicals were from Sigma (St. Louis, MO, USA).
All the human breast cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC1428, MDA-MB-231, BT549, HCC1937, HCC1569, HCC70, HCC1500, HS578T, SK-BR-3, AU565 and ZR-75-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-7, T47D, BT474, BT483, MDA-MB-468, MDA-MB-453, MDA-MB-435 and MDA-MB-361 cells were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. MCF-10A cells were cultured in DMEM/F12 supplemented with 20 ng/mL epidermal growth factor, 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, 5% heat-inactivated HS, 100 IU/mL penicillin and 100 μg streptomycin. All the cell lines were grown at 37°C in an atmosphere containing 5% CO2 and 95% air.
Gli1-specific shRNA inhibition
To knock down Gli1 expression, shRNA targeting Gli1 expressed in the pSingle vector was prepared as described previously. Cells were grown in culture dishes until they reached 75% confluence, at which point they were transfected for 24 h with pSingle-shRNA specific to Gli1 using the Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions. The tight on/off regulation of the pSingle vector system and coordinate inactivation of the target gene was mediated using doxycycline (Dox). Expression of the shRNA in the absence of induction was extremely low and prevented unwanted suppression of the target gene. When Dox was added to the culture medium, transcriptional suppression was relieved, permitting the shRNA to be transcribed. After transfection, cells were trypsinized, collected and subjected to various experiments.
Total RNA was extracted from different cell lines using the TRIzol reagent. Quantitative determination of RNA levels was performed in triplicate in three independent experiments. Real-time PCR and data collection were performed on the ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). The housekeeping gene GAPDH was used as an internal control to normalize the expression levels of different genes. Quantification of the relative expression of target genes was performed using the ΔΔCt method. The following gene-specific primers were used: ERα, forward 5′-AGA TGG TCA GTG CCT TGT TGG-3′ and reverse 5′-CCA AGA GCA AGT TAG GAG CAA ACA G-3′; Shh, forward 5′-GTG TAC TAC GAG TCC AAG GCA C-3′ and reverse 5′-AGG AAG TCG CTG TAG AGC AGC-3′; Gli1, forward 5′-GCG ATC TGT GAT GGA TGA GAT TCC C-3′ and reverse 5′-TGC CTT GTA CCC TCC TCC CGA A-3′; SOX2, forward 5′-GCT GTA TGG CTG CTG CAC TTC A-3′ and reverse 5′-GCA CAC GCA CCC AGC ACT GT-3′; Nanog, forward 5′-AAT ACC TCA GCC TCC AGC AGA TG-3′ and reverse 5′-TGC GTC ACA CCA TTG CTA TTC TTC-3′; Bmi-1, forward 5′-GAC CAC TAC TGA ATA TAA GG-3′ and reverse 5′-CAT TTG TCA GTC CAT CTC TC-3′; ALDH1, forward 5′-GTT AGC TGA TGC CGA CTT GG-3′ and reverse 5′-CCC ACT CTC AAT GAG GTC AAG-3′; Ptch1, forward 5′-TCG CTC TGG AGC AGA TTT CC-3′ and reverse 5′-TCT CGA GGT TCG CTG CTT TT -3′; and GAPDH, forward 5′-CAA GGT CAT CCA TGA CAA CTT TG-3′ and reverse 5′-GTC CAC CAC CCT GTT GCT GTA G-3′.
Western blot analysis
Cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl [pH 7.4], 1 mM EDTA, 1 mM EGTA, 2 mM NaF, 1 mM sodium orthovanadate, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 10 μg/mL aprotinin, 10 μg/mL E 64 and 1 mM Pefabloc; EMD). Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China) and then boiled for 5 min. Protein samples (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, MA, USA). Membranes were rinsed in Tris-buffered saline containing Tween 20 (TBST), blocked with 5% bovine serum albumin (BSA) for 2 h at room temperature, and incubated with the primary antibody at 4°C overnight. The membranes were then rinsed and incubated in peroxidase-conjugated secondary antibodies for 1 h at room temperature. After washing, proteins were detected using enhanced chemiluminescence (ECL) (Millipore Corporation). Membranes were stripped and reprobed with anti-β-actin mouse monoclonal antibodies to confirm equal loading of samples.
Confocal immunofluorescence microscopy
Cell lines were plated on culture slides (Costar, Manassas, VA, USA). After 4 days, cells were rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde in PBS, and permeabilized using 0.5% Triton X-100. Cells were then blocked for 30 min in 10% BSA (Sigma Aldrich, St. Louis, MO, USA) in PBS and then incubated with primary monoclonal antibodies in 10% BSA overnight at 4°C. After three washes in PBS, slides were incubated for 1 h in the dark with FITC-conjugated secondary goat anti-mouse or goat anti-rabbit antibodies (Invitrogen). After three additional washes, slides were stained with 4-,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich) for 5 min to visualize the nuclei and examined using a Carl Zeiss confocal imaging system (LSM 780; Carl Zeiss, Jena, Germany).
Fluorescence-activated cell sorting (FACS) analysis
Anti-CD44-APC and anti-CD24-PE antibodies used for FACS analysis were obtained from Biolegend (San Diego, CA, USA). Briefly, for each cell line, 1 × 106 cells were aliquoted into two tubes; tube 1 was stained with IgG isotype controls for APC and PE, and tube 2 was stained with anti-CD44-APC and anti-CD24-PE antibodies. Cells were incubated with the appropriate antibodies for 30 min on ice and then washed with PBS. Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences); each sample required 10,000 cells for analysis.
Cell cycle assays
Aliquots of 1 × 105 cells were collected using trypsinization and treated with 50 μg/mL DNase-free RNase and 20 μg/mL propidium iodide (PI) following the manufacturer’s instructions. Cells were analyzed using an FC500 instrument (Beckman Coulter, Brea, CA) with MultiCycle for Windows software (Beckman Coulter) for detailed cell cycle status.
Apoptosis was determined using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, cells were detached and resuspended in 100 μL binding buffer containing FITC-Annexin V and PI. After incubation for 15 min at room temperature in the dark, cells were analyzed using an FC500 instrument (Beckman Coulter). Annexin V-positive cells were classified as apoptotic.
Mammosphere culture was performed as described by Dontu et al. with slight modifications. Single-cell suspensions were plated in ultralow attachment 96-well plates (Costar) at different densities of viable cells. Cells were grown in serum-free mammary epithelial growth medium (MEGM), supplemented with 1:50 B27 (Invitrogen), 20 ng/mL epithelial growth factor (EGF), 20 ng/mL basic fibroblast growth factor (bFGF; BD) and 10 μg/mL heparin (Sigma). The number of spheroids was counted after 7–10 days. For in vitro propagation, primary spheres were collected, dissociated into single-cell suspensions and plated in ultralow attachment 96-well plates. The secondary number of spheroids was counted 14 days after plating.
Colony formation assay
Cells were seeded in triplicate at 500 cells/6-cm dish in complete medium. After 3 weeks of growth, cells were fixed and stained with crystal violet (0.1% w/v in 20 nM 4-morpholinepropanesulfonic acid; Sigma), and visible colonies were counted according to the number of cells in each colony. All experiments were repeated at least three times. Plating efficiency was determined as the number of colonies formed divided by the total number of cells plated.
Wound scratch migration assay
Cells were seeded in 6-cm culture dishes, and cell monolayers were wounded by scratching with sterile plastic 200-μL micropipette tips and photographed using phase-contrast microscopy immediately following and 48 h after wounding. Migration assays were independently performed in triplicate. The migration distance of each cell was measured after the photographs were converted to Photoshop files.
Matrigel invasion assay
Invasion of cells was measured in Matrigel (BD)-coated transwell inserts (6.5 mm, Costar) containing polycarbonate filters with 8-μm pores, as detailed previously. Inserts were coated with 50 μL of 1 mg/mL Matrigel matrix according to the manufacturer’s recommendations. A total of 2 × 105 cells in 200 μL serum-free medium were plated in the upper chamber, and 600 μL of medium containing 10% FBS was added to the lower chamber. After 24 h incubation, top cells (noninvasive) were removed, and bottom cells (invasive) were counted. Cells that invaded to the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. For each membrane, five random fields were counted at 10× magnification. Data were presented as the mean ± SD from three independent experiments performed in triplicate.
Chromatin immunoprecipitation (ChIP)-qPCR
A chromatin immunoprecipitation kit (Cat. 17–371) was purchased from Millipore and ChIP experiments were carried out essentially as described (27). Immunoprecipitated DNA was analyzed on an ABI PRISM 7900HT sequence detection system. Primers used for detection of promoters following ChIP are located at -653 to -472 bp (1), -297 to -116 bp (2) and +167 to +359 bp (3) of the Gli1 promoter.
Tissue microarrays and immunohistochemical analyses
Tissue microarrays of breast samples (BC081116a) were from Alenabio (Xian, China). The manufacturer provided clinical and pathological information. Immunostaining was performed using the avidin-biotin-peroxidase complex method (UltrasensitiveTM, MaiXin, Fuzhou, China). Sections were deparaffinized in xylene, rehydrated in a graded series of alcohols and then boiled in 0.01 M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with rabbit polyclonal anti-Gli1 antibodies (1:100 dilution), mouse monoclonal anti-ER antibodies (1:50 dilution) or mouse monoclonal anti-ALDH1 antibodies (1:100 dilution). Staining for these antibodies was performed at room temperature for 2 h. Biotinylated goat anti-mouse serum IgG was used as a secondary antibody. After washing, sections were incubated with streptavidin-biotin conjugated with horseradish peroxidase and the peroxidase reaction was developed with 3,30-diaminobenzidine tetrahydrochloride. Two independent, blinded investigators examined the slides randomly. Five views were examined per slide and 100 cells were observed per view at 400× magnification.
Data were described as the mean ± SD. Associations between protein expression levels in the breast tissue microarray were assessed using Spearman’s rank correlation test. Comparisons between different groups were carried out using the Student’s two-tailed t-test. The limit of statistical significance was set at a P-value <0.05. Statistical analysis was performed using SPSS/Win11.0 software (SPSS, Inc., Chicago, IL, USA).