Cell culture and transfection.
Human embryonic kidney cell line (HEK293T), human cervical epithelioid carcinoma cell line (HeLa) and non-small cell lung adenocarcinoma cancer cell line (H1975) were obtained from Dr. JunYing Yuan (Harvard medical school, Boston). HEK293T cells were cultured in DMEM (#SH30022.01, HyClone) supplemented with 10% FBS (#3682, Internegocios-sa) and 1% Penicillin /Streptomycin (#15140-122, GIBCO). H1975 cells were cultured in RPMI-1640 (#SH30809.01, HyClone) supplemented with 10% FBS and 1% Penicillin /Streptomycin. All cell lines were incubated at 37 °C with 5% CO2. For transfection, HEK293T cells were seeded in 6-well plates and transfected with indicated plasmids by using Lipofectamine 2000 (Invitrogen). MG-132 (SelleckChem) were used at a concentration of 20 µM for 6 h.
Reagents and antibodies.
MG-132 (#S2619) was purchased from SelleckChem, Ltd. The following antibodies were used: Anti-PD-L1 (#66248, HUABIO, WB 1:1000, IHC 1:200), anti-USP21 (#sc-515991, Santa Cruz Biotechnology, WB 1:200), anti-USP21 (#EM-1901-37, HUABIO, IHC 1:200), anti-Flag-HRP (#M185-7, MBL, WB 1:20000), anti-HA (#0906, HUABIO, WB 1:5000), anti-K48 (#05-1307, Milipore, WB 1:1000), anti-Ub (P4D1) (#SC-8017, Santa Cruz Biotechnology, WB 1:200), anti-β-tubulin (#M1305-2, HUABIO, WB 1:5000), goat anti-mouse IgG (H + L) secondary antibody, HRP (#31430, Thermo Fisher Scientific, 1:20000), goat anti-rabbit IgG (H + L) secondary antibody, HRP (#31460, Thermo Fisher Scientific, 1:20000).
Plasmids.
Full-length USP21 cDNA was provided by Life Sciences Institute, Zhejiang University. USP21 was cloned into pCMV3 by PCR amplifying from cDNA templates. USP21 C221A mutation was introduced by PCR and confirmed by DNA sequencing. The mutation primer sequences were: 5′-CCTGGGAAACACGGCCTTCCTGAATGCTG-3′ and 5′-CAGCATTCAGGAAGGCCGTGTTTCCCAGG − 3′. Full-length PD-L1 was provided by Life Sciences Institute, Zhejiang University. PD-L1 fragments 1–6 were PCR-amplified from pCMV3-3XFlag-PD-L1 plasmid. The primer sequences were: 1-F 5′-GGCATTTGCTGAACGCATTGGTAATTCTGGGAGCCAT-3′,1-R 5′-TGCGTTCAGCAAATGCCAGTAGGTCATGAA-3′; 2-F 5′-GGCATTTGCTGAACGCATAC AGACAGAGGGCCCGGCT-3′, 2-R 5′-TGCGTTCAGCAAATGCCAGTAGGTCATGAA-3′; 3-F 5′-AGGTTCAGCATAGTAGCCCCAAGGCCGAAGTCATCTG-3′,3-R 5′-GCTACTATGCTGAACCTTCAGGTCTTCCTC-3′;4-F 5′-GTCAGGCTGAGGGCTACTTGGTAATTCTGGGAGCCAT-3′, 4-R 5′-GTAGCCCTCAGCCTGACATGTCAGTTCATG-3′; 5-F 5′-CAAATGAAAGGACTCACAGAAAAGGGAGAATGATGGA-3′, 5-R 5′-GTGAGTCCTTTCATTTGGAGGATGTGCCAG-3′; 6-F 5′-CATTCATCTTCCGTTTATAATGATCTAGAGCGGCCGC-3′, 6-R 5′-TAAACGGAAGATGAATGTCAGTGCTACACC-3′. The PD-L1 mutation (K263R, K270R, K271R, K280R, K281R) was introduced by PCR and confirmed by DNA sequencing. The mutation primer sequences were: K263R-F: 5′-ATTCATCTTCCGTTTAAGAAGAGGGAGAATGATGGATGTGA-3′, K263R-R: 5′-TCACATCCATCATTCTCCCTCTTCTTAAACGGAAGATG AAT-3′; K270R-F: AGGGAGAATGATGGATGTGAGAAAATGTGGCATCCAAGATA-3′, K270R-R: TATCTTGGATGCCACATTTTCTCACATCCATCATTCTCCCT-3′; K271R-F: GAGAATGATGGATGTGAAAAGATGTGGCATCCAAGATACAA-3′,K271R-R: TTGTATCTTGGATGCCACATCTTTTCACATCCATCATTCTC-3′;K280R-F: CATCCAAGATACAAACTCAAGGAAGCAAAGTGATACACATT-3′, K280R-R: AATGTGTATCACTTTGCTTCCTTGAGTTTGTATCTTGGATG-3′; K281R-F: CCAAGATACAAACTCAAAGAGGCAAAGTGATACACATTTGG-3′, K281R-R: CCAAATGTGTATCACTTTGCCTCTTTGAGTTTGTATCTTGG-3′. The PD-L1 5K mutation primer sequences were: 5K-F: 5′-CATTCATCTTCCGTTTAAGAAGAGGGAGAATGATGGATGTGAGAAGATGTGGCATCCAA, 5K-R: 5′-TCCAAATGTGTATCACTTTGCCTCCTTGAGTTTGTATCTTGGATGCCACATCTTCTCACATCCA. The shRNA used to knock down USP21 expression of human was purchased from Vigene Biosciences, Inc. The shRNA target sequences for USP21 knockdown was 5′-GCTCCACCGATACTTGCCAATTTCAAGAGAATTGGCAAGTATCGGTGGAGCTTTTTT-3′.
Immunoblotting.
Samples were lysed in RIPA buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM NaF, 1 mM Na3VO4) and heated at 100 °C for 10 min in 2 × SDS loading buffer. Next, samples were separated by gel electrophoresis using 8–12% SDS-PAGE gels and transferred to nitrocellulose membranes for 1–2 h at 0.3A current on the ice. Membranes were blocked in 5–10% nonfat dried milk (Wandashan) diluted in PBS-T (PBS, 1% Tween-20) and probed with the indicated primary antibodies in PBST containing 5% (w/v) BSA (#9048-46-8, Sangon Biotech) overnight at 4 °C. All HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. All washing steps were performed using PBS-T (10 min, 3 times). Detection was performed using HRP-conjugated secondary antibodies and chemiluminescence reagents (#4AW001-500, Beijing 4A Biotech Co, Ltd.).
Immunoprecipitations.
HEK293T cells were transfected with indicated plasmids and collected in RIPA buffer at 4 °C for 30 min. After 15,000 g centrifugation at 4 °C, supernatant and following conjugated beads were combined and rotated at 4 °C for 6 h: anti-Flag (DYKDDDDK) affinity gel (#B23102, Bimake) and anti-HA magnetic (#B26202, Bimake) beads. The beads were collected and washed with RIPA buffer 4 times and heated at 100 °C for 10 min in 2 × SDS loading buffer prior to IB. HeLa cells were collected in RIPA buffer at 4 °C for 40 min. After 15,000 g centrifugation at 4 °C, supernatant from samples were pre-cleared to remove non-specific interactions with protein A/G beads rotating at 4 °C for 2 h. Following pre-clearing and PD-L1 antibody conjugation (4 °C for 2 h), lysate and beads were combined and rotated at 4 °C for 6 h. Following incubation, A/G beads were collected and washed with RIPA buffer 4 times and heated at 100 °C for 10 min in 2 × SDS loading buffer prior to IB.
In vitro deubiquitination assay.
HEK293T cells were transfected with Flag-PD-L1, HA-USP21, HA-USP21-C221A expression plasmids. Cells were collected 48 h post transfection following 6 h MG-132 (20 µM) treatment. Cells were lysed using a RIPA lysis buffer with protease inhibitor and centrifuged at 15,000 g for 10 min. The supernatant containing protein was incubated with Flag/HA-beads for 6 h. PD-L1 and USP21 were purified from the cell extracts with Flag/HA-beads in lysis buffer. For deubiquitination assay, ubiquitinated PD-L1 was incubated with HA-USP21 or HA-USP21-C221A in DUB Buffer (50 mM Tris pH 7.5, 50 mM NaCl, 5 mM DTT, 5 mM MgCl2). Reactions were incubated at 30 °C for 2 h on rotary shaker. Next, proteins were eluted from beads and heated at 100 °C. Ubiquitination status of PD-L1 was assessed by IB as described above.
Immunohistochemistry.
The IHC of human tumor samples was done by HUABIO, China, with mouse anti-PD-L1(#66248, HUABIO, IHC 1:200) and mouse anti-USP21 antibody (#EM-1901-37, HUABIO, IHC 1:200) imaging with OLYMPUS BX61 microscopy.
Clinical samples.
The Lung cancer samples were acquired from The First Affiliated Hospital, College of Medicine, Zhejiang University. Tissue samples were rapidly frozen in liquid nitrogen after surgical resection and stored at − 80 °C for further analysis.
Bioinformatics analysis.
Cancer genomics–related data were obtained from The Cancer Genome Atlas (TCGA, http://cancergenome.nih.gov) and Catalogue of Somatic Mutations in Cancer (https://cancer.sanger.ac.uk/cosmic) databases. In the result analysis, no sample was eliminated.
Statistics.
Statistical analyses were performed using the two-tailed Student’s t-test. Data were analyzed using the Prism software (GraphPad, San Diego, CA, USA) and expressed as the mean ± S.D. p < 0.05 was considered to be significant.