Cell lines and plasmids
BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; #11995-065; Gibco, Grand Island, NY, USA). The NSCLC cell lines A549, H1299, H1975, HCC827, H2170, H226, and H520 were obtained from ATCC (Manassas, VA, USA). H1299, H1975, HCC827, H2170, H226, and H520 cells were cultured in RPMI 1640 (#R8768; Sigma-Aldrich, Milwaukee, WI, USA), and A549 cells were cultured in Ham’s F-12K (#21127-022; Gibco, Grand Island, NY, USA). Media were supplemented with 10% fetal bovine serum (FBS; #10437-028, Gibco). The CRTAC1 overexpression plasmid and the vector plasmid were purchased from Sunny Biotechnology (Shanghai, China). The different siRNAs targeting CRTAC1 and its scrambled control siRNA were purchased from Jinweizhi Biotechnology (Suzhou, China). The Akt1 overexpression plasmid and control plasmid were purchased from Miaoling Biotechnology (Wuhan, China). The STUB1 shRNA plasmid, the control plasmid, and the STUB1 promoter plasmid (full length, from −1020 to +1) were synthesized by Qingke Biotechnology (Beijing, China). The STUB1 promoter Mut plasmid was constructed by the author. Point mutation PCR was performed using the forward outside primers (F1) 5′-CTATCGATAGGTACCAGGGAGGCCCCGCCC CCA CT-3′, and (F2) 5′-TGAGGCATCTCACTGCCTTTGTCGAATGTGTGTGG-3′, and the reverse primers (R1) 5′-ATCGCAGATCTCGAGAGCTCCGCCGGA-3′, and (R2) 5′-CACACACATTCGACAAAGGCAGTGAGATGCCTCAC-3′. The PCR products were digested and cloned into pGL3-Basic (E1751; Promega, Madison, WI, USA) and verified by DNA sequencing.
Cell transfection and generation of stable cell lines
Plasmids were introduced into cells using PolyjetTM DNA in Vitro Transfection Reagent (SignaGen Laboratories). GFP-CRTAC1 stable expression cells and vector control cells were constructed by lentivirus infection and screened by puromycin. Lentivirus packaging and infection experiments were performed as described previously . RiboFECT™ CP diluted in OptiMEM (Life Technology) was used to deliver the siRNA #1, 5′-GCAGUGCCUCGGAUAUCUUTT-3′, #2, 5′-GCCAAUU ACGCCUACGGUATT-3′, #3, 5′-UGGACCCAACCUGGUUCUGAATT-3′ targeting CRTAC1 into the cells according to the manufacturer’s instructions. The shRNAs targeting STUB1 were cloned into pLKO.1 and transfected into cells, then select stable transfection cells with G418 (Goldbio) for at least 3 weeks. The following three target sequences of STUB1 were used: shRNA-1, 5′-CCGGGAAGAGGAAGAAGCGAGACATCTCGAGATGTCTCGCTTCTTCCTCTT CTTTTTG-3′, shRNA-2, 5′-CCGGGCAGTCTGTGAAGGCGCACTTCTCGAGT AACTTTGAAAGAGGGAGCTTTTTTG 3′ shRNA-3, 5′-CCGGCGCGAAGAAG AAGCGCTGGAACTCGAGTTCCAGCGCTTCTTCTTCGCGTTTTTG 3′.
Reagents and antibodies
Cisplatin (15663-27-1) was purchased from Sigma-Aldrich. Cycloheximide and MG132 were from Santa Cruz Biotechnology (Dallas, TX, USA). BAPTA-AM (S7534) was from Selleck Chemicals (Houston, TX, USA). Dantrolene (S80373) was obtained from Yuanye Bio-technology Co., Ltd (Shanghai, China). Antibody against CRTAC1 (Abcam Cat# ab102548, RRID: AB_10710442) was purchased from Abcam (Cambridge, UK). Antibodies against cleaved caspase3 (9661S), Akt (pan) (4685S), Akt1 (75692S), p-Akt (ser473) (4060S), and STUB1 (2080S) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against MUL1 (Proteintech Cat# 16133-1-AP, RRID: AB_2147111), NFATC1 (66963-1-Ig) and β-actin (HRP-60008) were from Proteintech (Wuhan, China).
The gene expression data and Kaplan–Meier survival plots for NSCLC patients were obtained from the GEO (Gene Expression Omnibus (GEO), RRID:SCR_005012) database (GSE29013 and GSE14814). The 4D label-free quantitative proteomics technology (PTM-Biolab, Hangzhou, China) was used to analyze the mass spectra of the samples and construct the specific protein database. The identified proteins were annotated in the KEGG (KEGG, RRID:SCR_012773) database and enriched by Fisher’s exact test.
Western blot analysis
Cells were lysed in lysis buffer, and proteins were extracted. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were probed with the indicated primary antibodies, followed by incubation with AP-conjugated secondary antibodies. An enhanced ECF chemifluorescence system was used to detect the signals, and images were acquired on a phosphorimager (Typhoon FLA 7000; GE Healthcare, MA, USA).
Cell viability assay
Cell viability was examined with the CellTiter-Glo® luminescent cell viability assay kit (G7572, Promega). Cells (3 × 103 cells/well) were seeded in 96-well plates with 200 μl of RPMI 1640 medium containing 10% FBS. Then, the cells were exposed to cisplatin at different concentrations for 48 h. Next, 25 μl CellTiter-Glo® reagent and 25 μl PBS were added per well on a vortex for 2 min. After incubating for 10 min to stabilize the luminescence signal, the plates were analyzed using a microplate luminous detector (LB960, Berthold, Germany).
Cell apoptosis was determined using a cellular apoptosis assay kit (AP105, Lianke Bio). Cells were treated with cisplatin for 48 h and resuspended in a binding buffer. Then, 5 μl Annexin V-APC and 10 μl 7-AAD were added sequentially and incubated for 5 min in the dark. The apoptosis rate was detected by flow cytometry (BECKMAN, USA).
Nude mouse xenograft model
Animal experiments were performed at the animal institute of Wenzhou Medical University according to protocols approved by the Laboratory Animal Center of Wenzhou Medical University and the Laboratory Animal Ethics Committee of Wenzhou Medical University. Twenty-four female BALB/c athymic nude mice (3–4 weeks old) were obtained from GemPharmatech (GemPharmatech, RRID:SCR_017239; license number: SCXK 2018-0008; Nanjing, Jiangsu, China). After 1 week of growth, the nude mice were randomly divided into two groups, with 12 mice in each group. A total of 5 × 106 H1299 Vector cells or H1299 CRTAC1 cells/100 µl PBS were injected subcutaneously into nude mice. When the subcutaneous tumors were palpable on day 7 after injection, the mice were randomly divided into two subgroups (n = 6), a CDDP group and a vehicle control group. The CDDP group was treated intraperitoneally with 3 mg/kg cisplatin every 3 days, and the vehicle group was treated intraperitoneally with PBS. After 30 days of treatment, the subcutaneous tumor tissues of nude mice were obtained.
Tissue samples were embedded in paraffin after gradient dehydration, and 4 μM slices were made. After xylene dewaxing and alcohol gradient rehydration (100%, 95%, 90%, 80%, 70%, and 50%), microwave antigen retrieval was performed in citrate buffer. After cooling to room temperature, the samples were treated with 3% H2O2 for 10 min and incubated for 30 min with 3% fetal bovine serum at room temperature. The tissue samples were then incubated overnight at 4 °C with antibodies against CRTAC1 (GTX119558, GeneTex), MKI67 (Ab16667, Abcam), and cleaved caspase3 (9661S, Cell Signaling Technology). Staining was performed with the Ready-to-Use SABC-POD Kit (SA1022; BOSTER Biological Technology, Wuhan, China). Immunostained images were acquired on a Nikon Eclipse Ni microsystem (DS-Ri2).
Total RNA was isolated from cultured cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed into total cDNA using the PrimeScriptTM RT kit (#RR036A; TaKaRa, Kyoto, Japan) and then subjected to RT-PCR. mRNA expression levels were determined by qPCR on a Q6 real-time PCR System (Applied Biosystems, Carlsbad, CA, USA) with SYBR qPCR Master Mix (4309155, Applied Biosystems) using GAPDH as an internal loading control. The primers used in this study were as follows: human Akt1 (forward: 5′-CTTGCTTTCAGGGCTGCTCA-3′ and reverse: 5′-TACACGTGCTGCCACAGGA TAC-3′); human STUB1 (forward: 5′-CTCTCACGCTCCGCGGCAAT-3′ and reverse: 5′-GCCAAGGAGCAGCGGCTGAA-3′); and human GAPDH (forward: 5′-GACTC ATGACCACAGTCCATGC-3′ and reverse: 5′-CAGGTCAGGTCCACCACTGA-3′).
Luciferase reporter assay
Luciferase reporters driven by the STUB1 promoter (P-STUB1-WT and P-STUB1-Mut) and their control plasmid were co-transfected with pRL-TK (RRID: Addgene_11313) into cells. The relative luciferase activity of specific samples was evaluated using the Dual-Luciferase Reporter Assay System (E1500, Promega) and measured using a microplate luminous detector.
The SimpleChIP Enzymatic Chromatin IP Kit (9003, Cell Signaling Technology) was used for the ChIP assay. Briefly, 4 × 106 H1299 cells for each sample were prepared. Normal rabbit IgG and NFATC1 antibodies were used in the control and experimental groups, respectively. Finally, the levels of the STUB1 promoter in the input and IP samples were detected by PCR, with primers as STUB1-122bp-Forword: 5′-AGGTGGTCGGGACAGGCTGTT -3′ and STUB1-122bp-Reverse: 5′-CTGCTGA GGTCTCTTCGGAA-3′.
Nucleo-plasmic separation assay
The Thermo Fisher PARIS™ Kit was used to perform nucleo-plasmic separation experiments. Briefly, 5 × 105 vector control cells and CRTAC1-overexpressed cells were seeded into a 10 cm dish. Upon adhesion, cells were starved with RPMI 1640 medium containing 0.1% FBS for 12 h, and then the medium was switched to the complete medium with or without 20 μM cisplatin for 48 h. After being treated with trypsinization, cells were collected, resuspended using 500 μl of ice-cold cell fractionation buffer, and then incubated on ice for 5–10 min. Upon incubation, samples were centrifuged at 4 °C and 500×g for 5 min, and the supernatant of them was collected as the cytoplasmic component. The pellet was then resuspended with 500 μl of ice-cold cell disruption buffer and incubated on ice for 5–10 min to obtain nuclei components.
Intracellular Ca2+ concentration [Ca2+] measurement
Cells were trypsinized into single cell suspensions and incubated with 500 μl HBSS with 5 μM Calbryte™ 630 AM (AAT Bioquest, Inc., Sunnyvale, CA, USA) for 1 h at room temperature, according to the manufacturer’s instructions. Then, cells were washed twice with HBSS to remove excess dye and analyzed using a CytoFLEX flow cytometer to measure fluorescence intensity.
Statistical analyses were performed using GraphPad Prism (GraphPad Prism, RRID:SCR_002798, version 7.0, Inc., La Jolla, CA, USA) software, and data were presented as the mean ± standard deviation. Differences between groups were analyzed by independent t-test, and P < 0.05 represented a statistically significant difference and indicated by *. Pearson correlation analysis was used to analyze the correlations between two groups, and the results were expressed by R. The value range of R was between −1 and +1. The R value of a negative correlation was −1 to 0, and 0 to +1 represented a positive correlation. Significant differences between the two groups were expressed by P < 0.05.