All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). There were grown according to standard protocols. Human lung cell lines IMR90 and WI38 and human MRC5 fibroblasts were cultured in MEM medium. There were used within 10 passages. Human lung cancer cell lines (HCC827, H23, H358, A549, H460, SK-MES-1, H1650, H1666, Calu-1, Calu-3, and H1299) were maintained with RPMI 1640 medium (Welgene, Seoul, Korea). All media were supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (100X Pen Strep solution, Invitrogen). All cells were cultured in a humidified incubator with 5% CO2 at 37 °C.
Tissue microarray and immunohistochemistry
Tissue microarray of lung cancer tissues was performed using a tissue microarray kit BS04116 (US Biomax, Inc.; Rockville, MD, USA). Clinical staging was assessed based on the AJCC cancer staging system (8th Edition). Immunohistochemistry was performed utilizing anti-SCARA3 antibodies (NBP1–32130, 1:100; Novaus, Centennial, CO, USA) according to the manufacturer’s protocol. Briefly, antigen retrieval was performed using 1X antigen retrieval buffer (pH 9.0; Abcam, Cambridge, England) in a cooling chamber (Biocare Medial, Pacheco, CA, USA). Sections were heated under pressure for 15 min, allowed to cool for 20 min, and equilibrated to ambient temperature under tap water. Endogenous peroxidase was blocked using 3% H2O2 solution before incubation with primary antibody at 4 °C overnight. Tissue sections were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature (RT) before visualization using DAB. Finally, sections were counterstained with Harris’s hematoxylin.
For total protein extraction, cells were lysed in ice-cold M-PER mammalian protein extraction reagent (78,501, Thermo Fisher, Pittsburg, PA, USA) with protease inhibitor (Complete mini, Roche, Darmstadt, Germany) on ice for 10 min. Cells were broken by sonication and subsequently centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was collected and protein concentration was determined with a Bradford protein assay. Proteins were separated by (6–12) % SDS-PAGE and transferred to polyvinylidene difluoride membranes (PALL life sciences, Washington, NY, USA). Membranes were blocked with blocking solution (5% skim milk in TBS-T (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20)) at RT for 1 h followed by incubation with primary antibodies at 4 °C overnight. The membrane was cleaved prior to hybridization with the antibody. Primary antibodies were anti-SCARA3 (1:1000, NBP2–13286, NOVUS), anti-β-catenin (1:1500, 8480, Cell Signaling, Danvers, MA, USA), anti-Vimentin (1:1500, 5741, Cell Signaling), anti-MMP-9 (1:1500, 2270, Cell signaling), anti-Cleaved PARP (1:1500, 5625, Cell Signaling), anti-Cleaved caspase3 (1:1500, 9661, Cell Signaling), anti-phospho-Akt (1:1500, 9271, Cell Signaling), anti-Akt (1:1500, 9272, Cell Signaling), anti-phospho-SAPK/JNK (1:1500, 9251, Cell Signaling), anti-JNK1 (1:1500, 44-690G, Thermo Fisher), anti-Bcl-xL(1:1500, 2764, Cell Signaling), anti-Bax (1:1500, 2772, Cell Signaling), anti-Noxa (1:1500, OP180, CALBIOCHEM, Darmstadt, Germany), anti-GAPDH (1:8000, SC-47724, Santa Cruz, Dallas, TX, USA), anti-Flag M2 (1:200, F1804, Sigma-Aldrich), and anti-Actin (1:8000, MAB1501, Merck Millipore, Darmstadt, Germany). Membranes were washed three times with TBS-T buffer and incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:4000, Jackson Laboratory, West Grove, PA, USA) at RT for 2 h. After rinsing with TBS-T buffer three times, membranes were treated with immobilon western chemiluminescent HRP substrate (P90720, Merck Millipore). Specific bands were visualized using a Luminescent image analyzer LAS-4000mini (Fujifilm Life Science, Stanford, CT, USA) to evaluate protein expression levels.
Bioinformatics analysis of RNA-Seq data in TCGA
The TCGA mRNA expression of discovery set was transformed into log2 scale. TCGA datasets contained survival data with clinical information. TCGA survival curves were visualized using UCSC Xena browser (https://xena.ucsc.edu/) and GraphPad Prism software version 8.0.
Quantitative real-time PCR
Total RNAs of cells cultured in 60 mm culture dishes for 24 h were isolated using Trizol (Invitrogen) and reverse transcribed into cDNAs using Reverse Transcriptase M-MLV (Takara, Mountain View, CA, USA) according to the manufacturer’s protocol. Real-time PCR analysis was performed using a SYBR green-based fluorescent method (SYBR premix Ex Taq kit, Takara) and an ABI 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with specific primers. Primers used for real-time PCR were as follows: GAPDH forward, 5′-TTC ACC ACC ATG GAG AAG GC-3′ and GAPDH reverse, 5′-GGC ATG GAC TGT GGT CAT GA-3′; SCARA3 forward, 5′-GAA TTG CAG GGA AGA CAG GG-3′ and SCARA3 reverse, 5′-GTA GAA GCT CTG GCT TCC TGG-3′. The quantity of SCARA3 transcripts was calculated based on the threshold cycle (Ct) using the ∆∆Ct method after normalization against the level of GAPDH as an internal control.
Generation of stable flag-SCARA3 cells
H1299 and A549 cells were transfected with SCARA3-pcDNA3.1+/C-(K) DYK (Flag) vector (GenScript; Piscataway, NJ, USA) or control- pcDNA3.1+/C-(K)DYK (Flag) vector using Lipofectamine 2000 (Invitrogen). At 24 h post transfection, cells were expanded 1:20 into complete media containing 0.3 mg/mL neomycin. Selection with neomycin was usually completed within 2 to 3 weeks. Clones stably overexpressing Flag-SCARA3 were confirmed by Western blot analysis.
Migration and invasion assays
Cellular migration of H1299-SCARA3 and H1299-control cells was determined using 24-well Transwell permeable Supports (3422, Corning, Kennebunk, ME, USA). Cellular potential for invasiveness was determined using 24-well Matrigel invasion chambers (354,480, Corning). Cells were seeded into upper inserts at 2 × 105 cells in 300 μL serum-free RPMI 1640. Outer wells were filled with 700 μL RPMI containing 10% FBS as chemoattractant. Cells were incubated at 37 °C with 5% CO2 for 16 to 20 h, stained with 1% Crystal violet for 5 min, and washed twice with PBS. Non-migrated or non-invading cells were removed by swabbing the top layer. Invasive cells were observed and photographed under an optical microscope in three random fields. Cells were counted using ImageJ software.
Immunofluorescence microscopic analysis
Cells were cultured for 24 h on cover slips coated with poly-L-lysine (Sigma–Aldrich, St. Louis, MO, US). These cells were washed with PBS, fixed in methanol for 5 min at RT, and incubated with blocking buffer (1% BSA in PBS). These cells were then incubated with anti-β-catenin antibody (1:200, 8480, Cell Signaling) and anti-Flag M2 (1:200, F1804, Sigma-Aldrich) at 4 °C overnight. After washing with PBS three times, cells were incubated with secondary antibody (Alexa Fluor 594 chicken anti-rabbit, 1:200, Invitrogen) in blocking buffer at RT for 2 h. Cells were then washed with PBS three times and mounted using Fluorescent Mounting Medium with DAPI (GBI Labs, Mukilto, WA, USA). Images were acquired using a Zeiss LSM Meta confocal microscope (Carl Zeiss, Weimar, Germany) with an LSM Meta software. Image contrast and brightness were adjusted using an LSM image browser.
Cell growth assay
Cell growth was analyzed with an EZ-Cytox cell viability assay kit (EZ-3000, Dogen, Seoul, Korea). Briefly, 1 × 106 cells in the presence or absence of 5 μM LY294002 (Sigma–Aldrich) were cultured in a 96-well plate for 24 to 72 h. After adding 10 μL of ten-fold solution, cells were incubated at 37 °C with 5% CO2 for 1 to 2 h. Cell growth rate was determined by measuring absorbance at 450 nm with a microplate reader at two time points (72 h and 24 h). It was calculated as OD450 at 72 h/OD450 at 24 h.
Tumor sphere formation assay
Single colony-dissociated cells were seeded into 24-well plates with ultra-low attachment surface (3473, Corning) and further incubated at 37 °C with 5% CO2 in a humidified incubator for 12 days (d). After cell images were collected with an optical microscope, tumor-sphere diameter was measured with an iSolution Lite software.
Tumor formation in nude mice
Mice used in this study were 5-week-old male BALB/c nude mice purchased from NARA Biotech (Seoul, Korea). They were housed in a pathogen free facility (SPF) and treated according to standard protocols and animal welfare regulations. Mice used in the experiment were supplied with sufficient water and feed in sterile cages. H1299 and A549 cells overexpressing SCARA3 or deficient in SCARA3 were harvested, resuspended in PBS, and then injected subcutaneously into the left and right flanks of the BALB/C nude mice (1 × 106 cells per flank, n = 5 mice per group). The size of a visible tumor was measured every 3 to 4 d using micrometer calipers. Tumor volumes were calculated with the following formula: volume = 0.5a × b2, where a and b were the larger and the smaller tumor diameters, respectively. Mice were humanely sacrificed at 12 weeks after injection. Primary tumors were excised, immediately weighed, and fixed in 4% paraformaldehyde. Significant differences between groups were assessed by two-tailed paired two-way ANOVA using GraphPad Prism (GraphPad Software Inc., CA, USA). Animal experiments were performed in accordance with the guidance Chosun University Institutional Animal Care and Use Committee. ARRIVE guidelines (http://arriveguidelines.org) were followed.
Cell viability assay
Cell viability was analyzed with an EZ-Cytox cell viability assay kit (EZ-3000, Dogen) utilizing 1 × 106 cultured cells treated with 30 μM cisplatin (Sigma–Aldrich) for 24 or 48 h. After adding 10 μL of ten-fold solution, cells were continuously incubated at 37 °C with 5% CO2 for 1 to 2 h. Cell viability was assessed with survival percent of each sample based on the OD450 ratio of before/after treatment with cisplatin (treated growth OD450/untreated growth OD450 × 100).
All statistical analyses were performed with Student’s t-test, two-way ANOVA test, and Mann–Whitney test using GraphPad Prism (GraphPad Software Inc., CA, USA). Survival curves were plotted with the Kaplan–Meier method. All data are presented as mean ± standard deviation (SD). Statistically significant differences are indicated as follows: *, p < 0.05; **, p < 0.001; and ***, p < 0.0001.