Detection of ACADL expression in NSCLC

A total of 515 profiles of lung adenocarcinoma (LUAD) and 59 paracancer samples, and 501 profiles of lung squamous cell carcinoma (LUSC) and 49 paracancer specimens were obtained from the Cancer Genome Atlas (TCGA) database. (https://portal.gdc.cancer.gov/) followed by being annotated with HUGO annotation file [30]. Differential expression analyses between LUAD and its paracancerous tissues, LUSC, and paracancerous tissues, as well as NSCLC and its paracancerous tissues, were performed using limma package in R software. And the differential expression of ACADL between the above groups was displayed using violin plot. Additionally, the role of ACADL in NSCLC survival was evaluated using the online tool called: Kaplan-Meier plotter (http://kmplot.com/analysis/), which incorporates mRNA‐seq and microarray profiles from different tumors, along with overall survival information collected from TCGA, Gene Expression Omnibus as well as Cancer Biomedical informatics Grid databases [31]. The protein expression of ACADL in LUAD, LUSC and normal lung tissues were investigated with The Human Pathology Atlas database (https://www.proteinatlas.org) which collects various proteins expression in major cancer and normal tissue types verified by immunohistochemistry (IHC) [32]. There is a total of 3 normal lung tissues, 10 LUSC samples, and 12 LUSC samples. And all the sections were evaluated and recorded as “Not detected”, “Low”, “Medium” and “High”. Here, the sections were quantified as: 1 = Not detected; 2 = Low; 2 = Medium and 4 = High.

Culture and treatment of NSCLC cells

The RPMI-1640 medium (Gibco, USA) containing with 10% fetal bovine serum (Gibco, USA) was utilized for culture of NSCLC cell lines H292, H1944, H1299 and A549 at a 37 °C incubator with 5% CO2 atmosphere. A lentiviral vector carrying the full-length open reading frame of ACADL was obtained from GenePharma Corporation (China) and was utilized to construct cell lines that stabilized overexpressing ACADL in H1299 and A549. Lentiviral transfection was performed according to the user guidelines with a multiplicity of infection as 20. In addition, for ACADL knock out (KO-ACADL) in H292 and H1944, a ACADL CRISPR/Cas9 KO plasmid, and its corresponding scrambled control plasmid (KO-SCR), designed and constructed by Genomeditech (Shanghai, China), were utilized for cell transfection with lipofectamine 2000 according to the manufacturer’s instructions. And a pcDNA 3.1 plasmid containing the full-length open reading frame of ACADL was obtained from GenePharma Corporation (China) and was utilized to rescues ACADL expression in KO-ACADL cells with lipofectamine 2000. Follow-up experiments were conducted 48 h after transfection. The YAP activator, XMU-MP-1 (1 mM, Selleckchem, USA), and an equal volume of DMSO were used to treat the transfected cell for 24 h before the follow-up assays.

Western blot

The western blot assay was performed following the previously reported method [33]. The protein samples were extracted with RIPA solution (Beyotime Institute of Biotechnology, China) supplemented with PMSF (1 mM, Beyotime Institute of Biotechnology, China). Nuclear proteins were isolated using the nuclear and cytoplasmic protein extraction kit according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, China). Primary antibodies used were as follows: ACADL (dilution: 1:1000, cat.no: HPA011990, Sigma-Aldrich, USA), P21 (dilution: 1:1,000, cat.no: 2947, CST, USA), P27 (dilution: 1:1,000, cat.no: 3698, CST, USA), Cyclin B1 (dilution: 1:1,000, cat.no: 4135, CST, USA), Cyclin D1 (dilution: 1:1,000, cat.no: 55,506, CST, USA). YAP (dilution: 1:1,000, cat.no: 14,074, CST, USA), YAP (Ser127) (dilution: 1:1,000, cat.no: 13,008, CST, USA), GAPDH (dilution: 1:5,000, cat.no: 60004-1-Ig, Proteintech, China), Histone H3 (dilution: 1:1000; cat.no: ab1791, Abcam, USA) β-actin (dilution: 1:1000, cat.no: 60,008, Proteintech, China). The HRP labeled anti-IgG antibody (dilution: 1:20,000, cat.no: ZB-2306; ZSGB-BIO, China) was used to combine the primary antibodies. The bands were visualized using the ECL reagent (WBKLS0500, Millipore) and captured using the Gel Dock™ XR + system with the ChemiDoc MP System (Bio-Rad). The quantitative analysis of the western blot assay was performed using ImageJ.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

According to the corresponding manufacturer’s instructions, the RNA samples of cells were harvested with Hipure Total RNA Mini Kit II (Magen, China), followed by transcription with PrimeScript RT Reagent Kit (TaKaRa, Japan). Subsequently, qPCR was implemented via the kit from SYBR Premix Ex Taq II (TaKaRa, Japan). The cycling conditions were set as follows: Preheating at 95˚C for 10 min, followed by 45 cycles at 95˚C for 15 s, 60˚C for 30 s, and 72˚C for 30 s. The primer sequences for the target genes were provided in Table 1. The expression of target genes was normalized to GADPH with 2−ΔΔct formula.

Table 1 Primers sequences used in the RT-qPCR experimentsIHC and immunofluorescence (IF) staining

For IHC stating, antigen retrieval was performed by incubating the slices in citrate buffer at 100 °C for 3 min. Nonspecific epitopes were blocked with 3% BSA at room temperature for 30 min. Subsequently, the primary antibodies, including Ki67 (dilution: 1:200; ab15580, Abcam, USA), epithelial cadherin (E-cadherin) (dilution: 1:500; cat.no: ab40772, Abcam, USA), and proliferating cell nuclear antigen (PCNA) (dilution: 1:100; cat.no: ab92552, Abcam, USA), were utilized for incubating the tissue slices at 4˚C overnight. The HRP labeled anti-IgG antibody (cat.no: ZB-2306; ZSGB-BIO, China) was then applied and incubated at room temperature for 1 h. Visualization and nuclei staining were achieved using DAB (ZSGB-BIO, China) and hematoxylin (Solarbio, China) for 3 and 15 min, respectively. Lastly, images were acquired using a light microscope (OLYMPUS, Japan). And the immunohistochemical intensity was analyzed based on the mean integrated optical density (mean IOD) for expression of target proteins by Image J.

For IF staining, the cells inoculated in a 24-well plate were fixed with 95% ethanol for 20 min, permeabilizated by 0.2% Triton X-100 for 30 min and blocked with 3% BSA for 30 min. Next, the cells were incubated with the YAP antibody (dilution: 1:200, cat.no: 14,074, CST, USA) overnight at 4 °C. After removing the primary antibody, the cells were treated with IgG H&L antibody (cat.no: ab150079, Abcam, USA) for 1 h at room temperature. Finally, the nucleus was stained with DAPI (Solarbio, China) and observed under a fluorescence microscope (OLYMPUS, Japan).

Apoptosis detection

The apoptosis level of cells was evaluated with Annexin V-FITC Apoptosis Detection Kit (Sigma, USA) according to the official protocols. the cells were subjected to flow cytometry (BD Pharmingen, USA) and analyzed by FlowJo software.

Colony formation assay

The cells with a density of 4 × 103 cells/well were cultured in the 6-well plate and continuously cultured for 14 days followed by being fixed with 4% paraformaldehyde (Biosharp, China). Subsequently, the colonies were visualized by incubating them with 0.1% crystal violet at room temperature for 30 min. Colonies consisting of more than 50 cells were imaged using a camera and analyzed by using ImageJ.

Cell proliferation assay

For assessing cellular proliferation, cells were plated in 96-well plates at a density of 5 × 103 cells/well and cultured for an additional 72 h. The cellular activity was evaluated using the 5-ethynyl-2’-deoxyuridine (EdU) assay kit (Solarbio, China) following the official protocol. The images were captured with fluorescent microscope (Leica, Germany) and analyzed using ImageJ software.

Transwell assay

The transwell assay was utilized to evaluate the migration ability of cells. Briefly, cells at a density of 1 × 105/well were seeded in the upper chambers (Coring, USA), which pre-coated with Matrigel (BD, USA), with serum‑free RPMI-1640 medium. The bottom chambers were loaded with 600 µl medium containing 10% FBS. After culture for 24 h, cells on the upper surface were scraped off with a cotton ball. And cells on the inner surface were fixed with 4% paraformaldehyde and stain with 0.1% crystal violet. The images were captured with a light microscope (OLYMPUS, Japan). Quantitative analysis of migrated cells was performed using ImageJ.

Tumor growth xenograft model

All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health of the United States. The experimental protocols were approved by the Experimental Animal Ethics Committee of Anhui Medical University (grant.no: LLSC20211508). Male nude mice aged 6–8 weeks old (18∼22 g) were obtained from the Experimental Animal Center of Anhui Medical University. The mice were randomly divided into two groups: Control group (n = 3) in which the mice received NSCLC cells without any treatment and ACADL group (n = 6) that mice received NSCLC cells overexpressing ACADL. A total of 5 × 106 cells were suspended in 100 µl PBS and inoculated subcutaneously into the right flank of each mice. After 5 days of inoculation, three mice in the ACADL groups were randomly selected for intraperitoneal injection of XMU-MP-1 (1 mg/kg) twice a week for three weeks, while the remaining three were treated with an equal amount of PBS. The mice were monitored and measured every 5 days, and no deaths were observed during this study. Humane endpoints were established to prevent pain or distress in mice, and euthanasia was performed when the tumor volume exceeded 4.2 cm3 or when the mice lost more than 20% of their body weight within 1–2 weeks [34]. After 25 days of observation, the subcutaneous tumors were harvested by cervical dislocation under anesthesia with an intraperitoneal injection of 60 mg/kg pentobarbital sodium. Tumor volume was calculated using the formula: volumes = width2 × length/2.

Statistical analysis

Statistical analyses were performed using SPSS software (version 16; SPSS). Data are presented as mean ± S.D. from at least three independent experiments. The comparisons between two groups were conducted with unpaired Student’s t‑test. And the comparisons among three or more groups, one‑way ANOVA or two‑way ANOVA analysis followed by Tukey’s multiple comparisons post hoc test was conducted. P < 0.05 was considered statistically significant.