Cell culture and transfection

The LUAD cell lines NCI-H1944, NCI-H1650, NCI-H1437, NCI-H1395, and NCI-H1299 were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in a 37 °C incubator with 5% CO2. The cells were seeded in 24-well plates at a density of 5 × 104 cells per well and incubated overnight. OPN3 knockdown and overexpression plasmids, along with their respective control vectors, were obtained from Genechem (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For OPN3 overexpression in NCI-H1299 cells, adenoviral vectors (Ad-OPN3, MOI 1:100) were employed along with polybrene (Millipore Sigma Aldrich Corporation, 107689, final concentration 8 ng/mL). OPN3 knockdown was achieved using mouse OPN3-specific siRNA, with non-specific siRNA serving as a negative control.

Tumor formation assay in nude mice

Female BALB/c nude mice, aged 4 to 5 weeks, were housed in a pathogen-free facility. NCI-H1437 cells, transfected with either shNC or shOPN3, were harvested, washed, and resuspended in phosphate-buffered saline (PBS). A 150 μL suspension containing 5 × 106 cells was subcutaneously injected into the left inguinal region of each mouse, with three mice per experimental group. Thirty-five days post-injection, the mice were euthanized, and the tumors were surgically removed for further analysis. Tumor images were captured, weights recorded, and volumes determined using the formula: (length × width2) × 0.5.

RNA extraction and real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, 15596026), and cDNA synthesis was carried out using the PrimeScript reverse transcription kit, following the manufacturer’s instructions. Real-time PCR was performed with the SYBR Green PCR Kit (Applied Biosystems, 4309155). Gene expression was quantified by calculating the Ct (threshold cycle) values and normalizing them to GAPDH levels. All samples were processed in triplicate. The primer sequences for OPN3 were sourced from Jiao et al. [19].

Western blot analysis

Cells were lysed in ice-cold RIPA buffer (Solarbio, R0010) supplemented with protease and phosphatase inhibitors (Abcam, GR304037-28). Protein concentrations were determined using the BCA protein assay kit. Proteins were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, and blocked with 10% non-fat milk. Primary antibodies against Phosphorylated Nuclear Factor Kappa B (P-NF-κB), Nuclear Factor Kappa B (NF-κB), CyclinB1, OPN3, Phosphorylated Extracellular Signal-Regulated Kinase (p-ERK), Extracellular Signal-Regulated Kinase (ERK), and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) were incubated overnight at 4 °C. Following incubation with appropriate secondary antibodies, bands were visualized and analyzed using Bio-Rad’s Image Lab 3.0 software. Anti-GAPDH antibody (Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436) served as the loading control.

Cell counting kit 8 (CCK8) assay

After OPN3 knockdown or overexpression, cells were seeded in 96-well plates at a density of 1 × 103 cells per well. CCK8 (Vazyme) was added at 24-h intervals for 4 days, and optical density at 450 nm was measured using a Bio-Rad iMark plate reader following a 2-h incubation.

Transwell assay

The migratory and invasive capabilities of H1437 cells were assessed using 24-well polyester membrane inserts (pore size: 8.0 μm, JET, Guangzhou, China). Cells were seeded in the upper chamber in serum-free medium at densities of 2 × 104 or 4 × 104 cells per well. In invasion assays, the upper chamber was pre-coated with Matrigel. After 16 to 24 h, cells that had migrated to the lower chamber were fixed with methanol and stained with 0.5% crystal violet. Migrating or invading cells were quantified microscopically.

Plate clone formation assay

OPN3 knockdown or overexpressing cells were seeded into plates at densities of 500 or 10,000 cells per well. After 14 days of incubation, cells were fixed with 4% paraformaldehyde (PFA) and stained with Giemsa stain (Leagene). Colony formation was then quantified and analyzed.

TUNEL staining

Apoptotic cells were assessed using the TUNEL assay kit (MA 0224, Meilun Biology, China). After fixation with 4% PFA, cells were permeabilized with 0.1% Triton X-100. The TUNEL reaction mixture was applied, and the cells were incubated at 37 °C for 60 min. DAPI was used for nuclear counterstaining, and apoptosis was visualized using fluorescence microscopy.

Flow cytometry

NCI-H1437 and siOPN3-H1437 cells were washed with PBS, subjected to trypsinization, and then collected by centrifugation at 1000 × g for 5 min. The cells were resuspended in PBS, and 2 × 105 cells were incubated with 195 μL of Annexin V-FITC binding solution. After treatment with Annexin V-FITC and propidium iodide staining solutions, the cells were gently mixed and incubated in the dark for 20 min at 37 °C. Apoptosis was evaluated by flow cytometry.

Wound healing assay

Cells (1 × 105) were seeded in 6-well plates, scratched with a pipette tip, and washed three times with PBS. The wound area was monitored, and images were captured and analyzed using a microscope.

Immunohistochemistry

Two days after transfection, cells were harvested and transferred to laser confocal dishes for a 12-h incubation, followed by two washes with PBS. The cells were then fixed in 4% PFA containing 2 mg/mL glycine for 15 min and permeabilized with 0.2% Triton X-100 for 5 min. After blocking with 5% BSA for 30 min, the cells were incubated overnight at 4 °C with anti-OPN3 primary antibodies. Subsequently, the cells were treated with Alexa Fluor-conjugated secondary antibodies (Earthox, Millbrae, CA, USA) and DAPI (Beyotime, Biotechnology).

H&E staining

For H&E staining, lung tissue was extracted from mice, washed with 1 × PBS, and fixed overnight in 4% PFA. The tissue was then embedded in paraffin, sectioned into 4 μm-thick slices, and stained with H&E. Images were captured using an optical microscope (Olympus, Tokyo, Japan).

Statistical analysis

OPN3 expression across various human cancers was assessed using data from the TCGA database. Protein expression of OPN3 in LUAD was determined through IHC staining results from the Human Protein Atlas (HPA) database. The relationship between OPN3 expression and LUAD progression stages was analyzed via the GEPIA2 platform. Each experiment was performed in triplicate, with results presented as mean ± standard deviation. Statistical analysis involved an independent two-sample t-test to compare means between two groups and one-way ANOVA to assess differences among three or more groups. Statistical significance was denoted as * for P < 0.05, ** for P < 0.01, *** for P < 0.001, and **** for P < 0.0001.