Human Tissues samples
A total of 30 pairs of lung cancer tissues and adjacent-matched normal tissues were used to examine mRNA levels and protein levels of different genes.
Cell culture
Cell lines (A549, H358, H446, H1299 and 16HBE) were obtained from cell bank of Shanghai Biology Institute (Shanghai (P.R. China)). Cells were cultured in DMEM medium (Trueline, Kaukauna (USA)) supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM L-glutamine and 1% penicillin/streptomycin (Solarbio, Beijing (P.R. China)) and maintained under 5% CO2 atmosphere at 37 °C. The AKT inhibitor LY294002 (25 µmol/L; S1105, Selleck, USA) was dissolved in DMSO (D2650, Sigma USA) and added to cell culture.
Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)
RNAs were isolated using TRIzol Reagent (Invitrogen, Waltham (USA)). cDNA was synthesized by using cDNA synthesis kit (Thermo Fisher Scientific, Waltham (USA)) following the manufacturer’s specifications. The thermal cycle was set as follows: 95 °C for 10 minutes followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 45 seconds. All data represent an average of three replicates. Primers used were as follows: USP46: F 5'-AGAAGCCCAGAAAAGGATGAGG-3', R 5'-CAAAAGCCAGAAGCCGTGAC-3'; PHLPP1: F 5' -AATGTGCCTGAGTGGGTATGTG-3', R 5'-TCATCAGAAGGTTAGGTGGGAG-3'; Beta-actin: F 5'-CGTGGACATCCGCAAAGAC-3', R 5'-TGCTGGGAGCCAGAGCAG-3'.
Western Blotting (WB)
Whole protein lysates were extracted using Radioimmunoprecipitation assay (RIPA) lysis buffer (JRDUN, Shanghai (P.R. China)) containing EDTA-free Protease inhibitor Cocktail (Roche, Heidelberg (Germany)), followed by protein concentration measurement by an enhanced BCA protein assay kit (Thermo Fisher Scientific). 25 µg of proteins from each sample were ran in 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore, Billerica (USA)) overnight. After blocking with 5% nonfat dry milk for 1 hour at room temperature, the membranes were probed at 4 °C overnight with primary antibodies followed by secondary anti-mouse IgG antibody (1:1,000; Beyotime, Shanghai (P.R. China)) for 1 hour at 37 °C. Protein expressions were visualized by the enhanced chemiluminescence system (Tanon, Shanghai (P.R. China)). Detailed information of primary antibodies are as follows: anti-USP46 (Ab88795, Abcam (UK)), anti-PHLPP1(Ab135957 Abcam (UK)), anti-p-AKT(#9271, CST (USA)), anti-AKT (#9272, CST (USA)), anti-β-actin (#4970, CST (USA)).
Knockdown and overexpression of USP46
Lentiviral plasmids (pLKO.1) containing three siRNAs directed to different regions of human USP46 (NM_001134223.2) and a negative control siRNA (siNC) were synthesized (Major, Shanghai (People’s Republic of China)). USP overexpression plasmid was constructed by inserting the full-length human USP46 cDNA sequence into Lentiviral plasmid (pLVX-puro) while the vector plasmid was used as a negative control (oeNC). Experiments were performed 48 hours after the transit transfection of the plasmids into lung cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instruction. Detailed nucleotide sequences are as follows: siUSP46-1: (8–26; TCCGAAACATCGCCTCCAT), siUSP46-2: (17–35; TCGCCTCCATCTGTAATAT), siUSP46-3 (26–44, TCTGTAATATGGGCACCAA).
Cell Proliferation assay
Cell proliferation was analyzed by Cell Counting Kit-8 (CCK-8) (SAB, College Park, (USA)) following manufacturer’s protocol. Briefly, cells were seeded in 96-well plates for 0, 24, 48 and 72 hours. CCK-8 solution (1:10) was added to each well and incubated for 1 hour. The absorbance was measured by the microplate reader (Pulangxin, Beijing (P.R. China)) at 450 nm.
Immunofluorescence (IF)
Cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. After that, samples were washed with PBS three times, 3 minutes each time, at 25 °C and blocked with 1% BSA (Solarbio, Beijing (People’s Republic of China)) for 1 hour at room temperature. Subsequently, cells were incubated with rabbit anti-γ-H2AX antibody (ab2893, abcam (UK)) in PBS overnight at 4 °C followed by goat anti-rabbit IgG (H + L) (A0423, Beyotime, Haimen (People’s Republic of China)) for 1 hour at room temperature. Images were obtained by an ECLIPSE Ni microscope and a digital image analyzer (NIKON, Tokyo, Japan).
Co-Immunoprecipitation (Co-IP)
For IP, whole-cell extracts were prepared after transfection or stimulation with appropriate ligands, the lysates were incubated overnight with the appropriate antibodies conjugated to Protein A/G beads (Santa Cruz Biotechnology, (USA)). Beads were washed five times and separated by western blotting as described above.
Ubiquitination Assay
After 48 hours of transfection with oeNC or oeUSP46, the cells were lysed with 1% SDS-containing RIPA buffer, and sonicated. Subsequently, samples were incubated with IgG (sc-2027, Santa Cruz Biotechnology (USA)) or PHLPP1 antibody (PA5-34434, Invitrogen (USA)) overnight at 4℃, followed by incubation with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology (USA)) for 1 hour. The beads were centrifuged at 3000 rpm for 5 minutes at 4 ℃ and subsequently washed with wash buffer for four times. The purified proteins were separated by 4–20% gradient SDS-PAGE protein gel, and immunoblotted with anti-ubiquitin antibody (ab7780, Abcam, UK).
Data analysis
Data were shown as mean ± standard deviation (SD) based on three independent experiments. Statistical significance was determined by one-way ANOVA for multiple comparisons using GraphPad Prism software version 7.0 (USA). P-value < 0.05 was defined as statistically significant.