Cell lines and cell culture
The human lung cancer cell lines A549, H1299, HCC827, H1975, H460, H1650 and embryonic kidney (HEK293) cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences Shanghai Institute of Biochemistry and Cell Biology. Cells were cultured in high-glucose DMEM (DMEM-HG) containing 10% FBS and were incubated at 37 °C in a humidified atmosphere with 5% CO2. All cell lines were authenticated by the DNA finger printing analysis and tested to be free of mycoplasma infection. These cell lines were Mycoplasma-free and routinely authenticated by quality examinations of morphology and the growth profile.
Western blot analysis
Total proteins extracted from the cells was separated by 8–12% SDS-PAGE and transferred onto nitrocellulose membranes, which were incubated with specific antibodies overnight at 4 °C, probed with HRP-conjugated secondary antibodies, and visualized using enhanced chemiluminescence reagent (Pierce). β-actin was used as the loading control. The following antibodies were used: TRIM15 (Cat: PA5-40946, Invitrogen), Keap1 (Cat: PA5-99434, Invitrogen), Nrf2 (Cat: PA5-27882, Invitrogen), anti-6x-His (Cat: MA1-21315, Invitrogen), anti-HA (Cat: 32-6700, Invitrogen), NQO1 (Cat: MA1-16672, Invitrogen), anti-K48-linkage Specific Polyubiquitin (Cat: #8081, Cell Signaling Technology), anti-K63-linkage Specific Polyubiquitin (Cat: #5621, Cell Signaling Technology).
A lung tissue microarray containing 98 cases of lung adenocarcinoma and paired adjacent non-cancerous tissue was purchased from Shanghai Outdo Biotech (HLugA180Su08). The incubated slides were then deparaffinized in xylene and rehydrated with graded alcohol. Next, antigens were retrieved using citrate buffer (pH 6.0). The samples were covered with 10% normal goat serum in phosphate buffered saline (PBS) for approximately 10 min at room temperature and then incubated with anti-TRIM15 (Catalog: PA5-40946, Invitrogen) or Nrf2 (Catalog: PA5-40946, Invitrogen) at 4 °C overnight. For immunohistochemical detection an HRP-Polymer Detection Kit (Abcam) followed by a DAB Substrate Kit (Abcam) were used, and slides were subsequently counterstained with haematoxylin. The staining intensity was scored as follows: 0 (0% to ≤ 25%), 1 (25% to ≤ 50%), 2 (50% to ≤ 75%), and 3 (> 75%). The 0 and 1 groups were defined as low expression, while 2 and 3 groups were defined as high expression.
Co-IP assays were performed as described previously. Briefly, cells were harvested in IP lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% TritonX-100, 1 mM ethylenediaminetetraacetic acid (EDTA), and protease inhibitors), incubated for 40 min on ice and centrifuged at 12,000×g at 4 °C for 10 min. Cell lysates were immunoprecipitated with antibodies against TRIM15 and Keap1 or negative control IgG and protein A/G agarose beads overnight with rotation. These beads were washed three times with lysis buffer. After separation by SDS-PAGE, the immunoprecipitates were subjected to Western blot analysis.
To detect the ubiquitination levels of Keap1, cells were transfected with or without indicated plasmids for different experimental purpose and treated with 10 μM MG132 for 4 h to block proteasomal degradation. The cells were lysed and immunoprecipitated for 4 h at 4 °C with Protein G Magnetic beads loaded or bound with anti-HA antibodies according to immunoprecipitation assay described above. The immunoprecipitated proteins were subjected to immunoblotting analysis with antibody against Ubiquitin.
CCK-8 cell proliferation assays
Cell viability was detected using the CCK8 assay (Dojindo, Tokyo, Japan). Briefly, the NSCLC cells stably transfected with shRNA were seeded into 96-well plates at a density of 4 × 103 cells per well. After 48 h, 10 μL of the CCK8 solution was added into each well. After an additional 4 h incubation, absorbance at 450 nm was measured using a microplate reader.
EdU incorporation assay
In the EdU assay, cells were plated in 12-well plates and transfected. After 48 h, EdU assay Kit (RiboBio Inc., Guangzhou, China) was performed to analyze cell proliferation per the manufacturer’s instructions. The cells were incubated with 10 μM EdU solution for 2 h and fixed with 4% paraformaldehyde, and the cells were washed 3 times with PBS and once with 0.5% TritonX-100. Next, the cell nuclei were stained with DAPI at a concentration of 1 µg/ml for 20 min. The proportion of the cells incorporated EdU was determined with fluorescence microscopy. After washing 3 times with PBS, we obtained images from a fluorescence microscope for further calculation of proliferation rates.
Total RNA was isolated from the cells using TRIzol Reagent (Thermo Scientific, MA). qPCR analysis was performed using standard procedures on a StepOnePlus Real-Time PCR System (Applied Biosystems, CA). PCR reactions were performed in triplicate and the relative amount of cDNA was calculated by the comparative CT method using RPS21 as an endogenous control. RT-PCR was performed in at least three biological replicates. The Taqman probe for NQO1 (Cat: HP101580), PRDX1 (Cat: HP101099), TXN (Cat: HP100418), FTL (Cat: HP104808), and GAPDH (Cat: HP100003) were purchased from Sino Biological Inc. (Beijing, China). The primers for Human GCLC: 5′-ATGTGGACACCCGATGCAGTATT-3′ (forward) and 5′-TGTCTTGCTTGTAGTCAGGATGGTTT-3′ (reverse) and HMOX: 5′-AACAAGCAGAACCCAGTCTATGC-3′ (forward) and 5′-AGGTAGCGGGTATATGCGTGGGCC-3′ (reverse). Gene expression was normalized to housekeeping gene expression (GAPDH).
Migration and invasion assays
Cells were allowed to form a confluent monolayer in a 24 well plate. The wound was created by scraping a conventional pipette tip across the monolayer. Cells were washed with PBS, cultured in serum and antibiotic free media at 37 °C, and photographed at 0 and 24 h.
For the in vitro invasion assays, cells in serum-free medium were seeded on a Matrigel-coated transwell insert (8 μm pore size, Corning, BD Biosciences). The lower chamber contained DMEM supplemented with 10% FBS as a chemoattractant. After incubation for 24 h, the inserts were fixed and stained for 30 min with 0.4% crystal violet dissolved in methyl alcohol, and the cell numbers were calculated by averaging the counts of three random fields.
Plasmid constructs and RNA interference
TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc. Scrambled, human TRIM15 and Nrf2 short hairpin RNAs (shRNAs) was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). To elucidate the role of TRIM15 as an E3 ligase, we generated an E3 ligase-defective TRIM15 by using the KOD-Plus-Mutagenesis kit (Toyobo, Cat: SMK-101) and verified by performing DNA sequencing. Plasmid transfection was performed using the Lipofectamine 3000 transfection reagent (Thermo Fisher, Cat. #L3000015) according to the manufacturer’s instructions. After 48 h, biological and biochemical experiments were performed.
Antioxidant response element (ARE) activity assays
The 8xARE-Luc construct was generated by cloning 8 copies of the antioxidant response element into the pGL3-promoter vector that contains the firefly luciferase gene. NSCLC cells were transfected with the 8xARE-Luc construct along with a renilla luciferase construct. 48 h after transfection, cells were subjected to overnight drug treatments and luciferase assays were performed using the Dual-Glo luciferase assay system. The firefly luciferase activity was, then, normalized to the renilla luciferase activity.
Immunofluorescence confocal imaging
Cells were grown on Lab-Tek chamber slides, fixed with 4% paraformaldehyde in PBS for 30 min, and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The slides were incubated with primary antibodies in blocking solution overnight at 4 °C in a humidified chamber. Subsequently, the glass slides were washed three times in PBS and incubated with Alexa Fluor 594-conjugated and Alexa Fluor 488-conjugated secondary antibodies and 4, 6-diamidino-2-phenylindole (DAPI) in blocking solution for 30 min at 37 °C in a humidified chamber. Images were acquired with a Leica confocal system.
Xenograft transplantation experiments
For subcutaneous xenografting, H1299 or H1650 cells were injected subcutaneously into 6-week-old male BALB/c nude mice. Tumors were measured twice weekly using calipers and the volume determined using the formula: V = (S2 × L)/2, where V is the volume, S is the shortest diameter, and L is the longest diameter. The mice were euthanized on day 30, and the tumor size and weight were measured. For the lung metastasis studies, H1299 or H1650 cells were injected into 6-week-old male BALB/c nude mice via the tail vein. Mice were sacrificed one months later, and the metastatic nodules were counted. Metastatic tissues were analyzed by hematoxylin and eosin staining. All murine procedures were approved by the Institutional Animal Care and Use Committee at Wannan Medical College.
Statistics were performed using GraphPad Prism 7 (Graph Pad Software Inc). The relationship of TRIM15 expression and clinicopathological parameters was evaluated with chi-squared test. The Kaplan–Meier method was used to analyse patient survival. Student’s t tests were used to evaluate continuous variables between subgroups. P value of less than 0.05 was considered statistically significant. All group numbers and explanation of significant values are presented within the figure legends.
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