br investigators provide evidence for the interaction of APP
investigators provide evidence for the interaction of APPBP2 with PPM1D. In short, this study's findings indicate that APPBP2 facilitates the oncogenesis of NSCLC through modulating the PPM1D and SPOP sig-nalling pathway.
2. Materials and methods
2.1. Cell culture and transfection
The immortalized lung adenocarcinoma cell line A549 (RRID: CVCL_0023), non-small cell lung cancer cell line H1299 (RRID: CVCL_0060), and human embryotic cell line HEK293T were purchased from the cell line bank of Bioon (Shanghai, China). Cell lines were cul-tured in Dulbecco's modified Eagle's medium (DMEM) with 10% foetal bovine serum (FBS, HyClone), 10 mg/ml streptomycin and 10u/ml pen-icillin. The cell lines were incubated at 37 °C in a 5% CO2 atmosphere.
2.2. Patients and tissue collection
Between September 2017 and October 2018, ten pairs of lung ade-nocarcinoma and adjacent normal lung tissues were collected at the First Affiliated Hospital of Bengbu Medical College (Anhui province, China) from patients who underwent surgical resection of lung cancer. The protocol of sample collection was approved by the Ethics Commit-tee of the First Affiliated Hospital of Bengbu Medical College (Bengbu, China). All patients provided informed consent for use of their samples in this study.
After fixing with 4% formalin and embedding in optimal cutting tem-perature compound (OCT), tissues were cut into 10 um sections using a microtome. Subsequently, the slides were treated with 3% H2O2 to block the endogenous peroxidase activity and heated (80 °C) for 10 min in cit-rate buffer (10 mM; pH 6·0) for antigen retrieval. To reduce nonspecific binding, 10% BSA + 0·4% triton-x100 was applied for 1 h at room tem-perature (RT). Subsequently, the slides were incubated with primary T16Ainh A01 (anti-APPBP2, Sigma, RRID: ab151305, 1:500; anti-PPM1D, Sigma, RRID: ab31270, 1:500; anti-SPOP, Abcam, RRID: ab168619, 1:500 at 4 °C overnight, followed by incubating with appropriate second antibodies. After that, DAPI (abcam, RRID: b104139) was applied for nu-cleus staining for 10 min at RT.
2.3.1. Extraction of total RNA, reverse transcription, and real-time polymer-ase chain reaction analysis
Total RNA was extracted from the cell lines and the paired tissue from ten patients with lung adenocarcinoma using Trizol reagent (Takara, 9108). Quality of the total RNA was determined using RNA gel electrophoresis and NanoDrop. Then, a total of 1 μg of high-quality total RNA were directly processed for synthesizing cDNA using PrimeScript™ RT reagent Kit (Takara, RR047A). The real-time polymer-ase chain reaction analyses were performed in a 20 μl volume system using SYBR (Takara, R820A, 10 μl) according to the manufacture's in-struction as follows: Primer mix (0·5 μl, 10 μM/each), template cDNA (1 μl), and sterile water (8·5 μl). The RT-PCR primers for APPBP2, PPM1D, SPOP, and GAPDH were summarized in Supplementary table 1.
Total protein from the human lung adenocarcinoma cancer samples and NSCLC cells were prepared with ice-cold radioimmuno-precipitation assay buffer, quantified using BCA protein assay reagent (Beyotime, P0009, China), and separated by SDS-PAGE. Following elec-trophoresis, proteins in the gel were transferred to polyvinylidene difluoride membranes (Merck Millipore). After blocking for 1 h with 5% non-fat milk, the membranes were incubated overnight at 4 °C
or antibody against β-actin (abcam, RRID: ab8226, 1:5000) which was set as the internal standard. This was followed by incubation with a horseradish peroxidase-labelled goat anti-rabbit IgG secondary anti-body (abcam, RRID: ab205718, 1:5000) or horseradish peroxidase-labelled goat anti-mouse IgG secondary antibody (abcam, RRID: ab6728, 1:5000) at room temperature for 1 h. Signals were detected with ECL Western Blotting Detection Reagent (GE Healthcare Life Sci-ences) and visualized using a Chemical Gel Documentation System (Tanon).
2.5. Gene silencing and overexpression
The knockdown of human APPBP2 was performed by shRNA . The investigators set sh-NC and sh-APPBP2 as the control and experimental groups, respectively. The target sequences of sh-NC and sh-APPBP2 are presented in supplementary table 1. sh-NC and sh-APPBP2 were cloned into pLKO.1 (Addgene,8453) plasmid, respectively. All the plasmids were sequenced. The investigators transfected shRNA-pLKO.1 with helper plasmids (VSVG and delta8·9) into HEK293T cells for 72 h to package lentivirus. The titer of the viruses was measured by examining the infectious ability of HEK293T cells. A549 and H1299 cells were in-fected with the sh-NC and sh-APPBP2 viruses. The investigators screened the stable transfected cell lines with 2 μg/ml puromycin, then RT-PCR and western blotting were applied for biological studies.