Analyzing ARNTL2 and ACOT7 in LUAD from TCGA

Gene Expression Profiling Interactive Analysis (GEPIA: http://gepia.cancer-pku.cn/) is a public database which analyzes the data from TCGA. We analyzed the expression of ARNTL2 and ACOT7, correlation between ARNTL2/ACOT7’s expression and patients’ survival, and the spearman correlation between ARNTL2 and ACOT7 in LUAD or LUSC patients based on TCGA database.

Cell culture

Human immortalized normal lung cells Beas-2B and NSCLC cells A549, H1299 and H1975 were purchased from ATCC and were grew in RPMI-1640 complete medium, in which 10% fetal calf serum and 1% antibiotics were added. The cells were maintained in a 37 °C cell incubator in which the CO2 concentration was kept at 5%.

RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

RNA was extracted from whole cell lysates using Trizol regent, following the manufacturers’ protocols. The RNA was reversely transcribed into cDNA using M-MLV reverse transcriptase (Promega). Quantification of the cDNA of indicated genes was conducted using SYBR master mixture (Takara) on Bio-rad real-time PCR system. The sequences of the primers were listed below: ARNTL2 forward, 5’-ACTTGGTGCTGGTAGTATTGGA-3’, and reverse, 5’-TGTTGGACTCGAATCATCAAGG-3’. ACOT7 forward, 5’-GGCCGGAAGCGGTATGAAG-3’, and reverse, 5’-GACTGGCTGTAGCTGACAGTG-3’. β-actin forward, 5’-GAGCTGCGTGTGGCTCCC-3’, and reverse, 5’-CCAGAGGCGTACAGGGATAGCA-3’.

Immunoblotting

To assess the protein abundance in cells, we collected whole cell lysates from indicated cells using RIPA lysis buffer (Beyotime). Protein concentration was determined by BCA assay kit (Beyotime). 30–50 μg of the total proteins were separated on SDS-PAGE gels and immunoblotted onto PVDF membranes, which were activated by methanol. To avoid non-specific protein signal, the membranes were incubated with 5% skim milk dissolved in 0.1% PBST (99.9% PBS and 1% TWEEN-20). After incubating with secondary antibodies at room temperature for 2 h, protein signals were detected using chemiluminescence assay kit, according to the manufacturers’ protocols. Primary antibody against ARNTL2 was from Sigma-Aldrich (SAB2100154). ACOT7 (15,972–1-AP) and GAPDH (60,004–1-Ig) antibodies were from Proteintech. Anti-mouse (sc-2005) and rabbit (sc-2004) secondary antibodies were from SantaCruz.

Cell proliferation and cell viability

Cell proliferation was analyzed by using CCK-8 assay kit (Beyotime). A549 cells transfected with siNC, siACOT7 (siACOT7#1 and siACOT7#2) or siARNTL2 (siARNTL2#1 and siARNTL2#2), and H1299 cells transfected with lenti-Ctrl, lenti-ACOT7 or lenti-ARNTL2, were seeded in 96-well plates at a density of 3000 cells per well. Each well contained 100 μl RPMI-1640 complete medium. For the indicated time, 10 μl of CCK-8 buffer was added in each well and the plates were maintained in the cell incubator for 3 h. After vibrating the plates for 10 min, OD450 was measured on a microplate reader. The siRNAs were obtained from Hippo Biotechnology (Huzhou, China) and the sequences were as following: siNC: 5’-UUCUCCGAACGUGUCACGU-3’; siARNTL2#1: 5’-GAUGGUGAACACCAAGUUA-3’; siARNTL2#2, 5’-GGACAAGACCAACAGCUAU-3’; siACOT7#1, 5’-GCAUGACCUUCACGAGCAA-3’; siACOT7#2, 5’-CGCUGAAGAAUGUGGACAA-3’.

For cell viability analysis, indicated cells were incubated with DMSO, ferrostatin-1 (ferr-1, 2 μM, Selleck), Z-VAD-FMK (Z-VAD, 8 μg/ml, Selleck), erastin (5 μM, Selleck), apoptosis activator 2 (AA2, 4 μM, Selleck) for 6–10 h and cell viability was tested by trypan blue staining. Cell death (%) = 100%—cell viability (%).

Colony formation

A total of 1000 siNC, siACOT7#1 and siACOT7#2 A549 cells and a total of 2000 siNC, siARNTL2#1 and siARNLT2#2 A549 cells were seeded in 6-well plates. A total of 500 lenti-Ctrl, lenti-ARNTL2 and lenti-ACOT7 H1299 cells were seeded in 6-well plates. 10 days later, colonies were formed. The plates were washed by PBS and the colonies were fixed by methanol and stained by crystal violet.

Cell cycle

A549 cells transfected with siNC and siACOT7 (siACOT7#1 and siACOT7#2), and H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus, were seeded in 6-well plates. 48 h later, cells were harvested and maintained in 70% cold alcohol for 8–12 h. Subsequently, cell cycle was analyzed by PI staining (YEASEN, 40301ES50) on flow cytometry.

Annexin V and PI staining

A549 cells transfected with siNC and siACOT7 (siACOT7#1 and siACOT7#2), and H1299 cells transfected with Ctrl and ACOT7 overexpression lentivirus, were seeded in 6-well plates. 48 h later, cells were harvested and subjected to PI/Annexin-V (YEASEN, 40302ES20) staining and analysis on flow cytometry.

Lipid ROS detection on flow cytometry

Resuspended cells were stained with 3uM of C-11 BODIPY dye (Invitrogen) for 30 min, according to the manufacturers’ protocols. Fluorescence intensity was analyzed on flow cytometry.

Caspase 3 to caspase 7 activity measurement

The activity of caspase 3 to caspase 7 was detected by using caspase-Glo reagent (Promega), according to the manufacturers’ protocols. In brief, after incubating the caspase-Glo reagent for 2 h, the activity of caspase 3 to caspase 7 was examined on a microplate reader.

Dual luciferase reporter activity

Dual luciferase reporter activity was analyzed by using assay kit from Promega, according to the manufacturers’ protocols. The coding sequence of ARNTL2 was inserted into pCDNA3.1 vectors. The promoter sequence of ACOT7 (-2000 ~ 0 bp) was inserted into pGL3.basic vectors. Dual luciferase reporter activity was examined 48 h after co-transfecting siRNAs (siNC or siARNTL2)/pCDNA3.1 (pCDNA3.1-Ctrl or pCDNA3.1-ARNTL2), pGL3.basic vectors and TK vectors into NSCLC cells. Relative luciferase activity was adjusted to TK activity.

Chromatin immunoprecipitation-qPCR assay

To assess whether ARNTL2 binds to the promoter sequence of ACOT7, we inserted the coding sequence of ARNTL2 into the pCDNA3.1-Flag plasmid. A549 and H1299 cells were transfected with the plasmids and subjected to chromatin immunoprecipitation assay by using SimpleChIP enzymatic chromatin IP kit (Cell Signaling), according to the manufacturer’s instructions. qPCR was applied to examine the DNA amplification samples in Chip-IgG and Chip-Flag group.

Fatty acid metabolism

The cellular abundance of MDA in siNC, siACOT7#1 and siACOT7#2 A549 cells, and in lenti-Ctrl and lenti-ACOT7 H1299 cells was measured by using the assay kit from Beyotime (Shanghai, China), according to the manufacturer’s instructions. The cellular abundance of triglyceride in A549 and H1299 cells was assessed by using the assay kit from Nanjing Jiancheng, according to the manufacturer’s instructions.

Statistical analysis

Statistical data were analyzed on GraphPad Prism 8 software and presented as mean ± standard error of mean (SEM). Students’t test was applied to determine the difference between two groups. One-way ANOVA followed by a Tukey’s post hoc test was used to determine the difference among groups. Statistical difference was considered significantly when p < 0.05.