Cell culture and establishment of stable transfected cell lines
Human MM cell lines LP-1, RPMI8226, KM-3 and SKO-007 were used in this study. LP-1 and SKO-007 cell lines were purchased from the American Type Culture Collection (ATCC). RPMI8226 cell line was kindly provided by Cell Bank Shanghai Institute of Cell Biology (Shanghai, China). KM-3 cell line was conserved in our laboratory. All MM cell lines were cultured in RPMI-1640 medium (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine (Gibco). The cells were grown at 37℃ in a humidified 5% CO2 atmosphere.
LP-1 cells were infected with lentivirus containing human PRAME RNA, PRAME shRNA lentiviral particles or corresponding blank control lentiviral particles (GeneChem, Shanghai, China). RPMI8226, KM-3 and SKO-007 cells were infected with human PRAME shRNA lentiviral particles or blank control lentiviral particles (GeneChem). Medium containing lentiviral particles was replaced with a complete medium after 12h of infection. Stably transfected cells were selected with 2 µg/mL puromycin dihydrochloride (GeneChem) at 96h post-infection for 2 weeks.
Rna Extraction And Qrt-pcr (Quantitative Real-time Pcr)
RNA was extracted and reverse-transcribed into complementary DNA (cDNA). PRAME transcript was measured by TaqMan-based qRT-PCR technology as we described previously[18]. The PRAME transcript level was calculated as PRAME copies/ABL copies in percentage.
Cell Proliferation Viable Assays
Cells were seeded into 12-well plates at a density of 1×104/mL. The evolution of cell number with time was followed 7 days by cell counting realized at successive time intervals of 24h. Experiments were performed in triplicate. In addition, the Cell-Light EdU Apollo643 In Vitro Kit (RioBio, Guangzhou, China) was used to measure cell proliferation according to the manufacturer’s instructions.
Cell Cycle Analysis
Cells were seeded in 12-well plates and starved by adding serum-free medium for G1 synchronization. After 24h, complete medium was added for an additional 48h. Cells were fixed in 75% ethanol, stained with prodium iodide (BD Pharmingen, San Jose, CA, USA). DNA content was measured by flow cytometry (BD biosciences, San Jose, CA, USA). The data were analyzed using Modfit LT2.0 software (Coulter Electronics, Hialeah, FL, USA).
Assessment Of Apoptosis
Cells were seeded in 12-well plates at 37℃ in a humidified 5% CO2 atmosphere for 48h with or without bortezomib. Apoptotic cells were determined using the Annexin V-APC/7-AAD apoptosis kit (KeyGEN BioTECH, Suzhou, China) according to the manufacturer’s instructions. Cells were immediately analyzed by flow cytometry (BD biosciences). Analysis was performed using Kaluza flow analysis software (Beckman Coulter, Brea, CA, USA).
Colony-forming Assays
Cells were suspended in 1ml of complete Methocult™ H4230 medium (STEMCELL Technologies, Vancouver, Canada) and plated in 6-well plates at a concentration of 1×104 cells/well. Colonies were maintained at 37℃ with 5% CO2 and 95% humidity for 12 days, Colonies of ≥ 50 cells were counted. Assays were performed in triplicate.
Cell Migration And Invasion Assay
Cell migration and invasion were evaluated using Transwell invasion assays with or without Matrigel (BD). 1×105 cells were seeded into the upper chamber of a Transwell insert (8 µm pores, Corning, NY, USA) in RPMI-1640. The upper chamber was placed into the Transwell containing a medium supplemented with 10% FBS in the lower chamber. The cells in lower chamber were counted after 24h and experiments were done in triplicate.
Xenograft Tumor Model
The animal studies were approved by the Institutional Animal Care and Use Committee of Peking University People's Hospital. Male BALB/c nude mice that were 4 to 6 weeks old were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were pretreated with intraperitoneal injections of cyclophosphamide once daily at 100 mg/kg for two consecutive days. The mice were randomly divided into two groups (n = 5), and LP-1 cells transfected with human PRAME shRNA lentiviral particles or blank control lentiviral particles were injected subcutaneously into the right forelimb armpit. Tumor diameters were measured every 2 days until day 30. Tumor volume (mm3) was estimated by measuring the longest and shortest diameter of the tumor as described[19]. Mice were euthanized on day 30 and tumors surgically removed for subsequent experiments.
Immunohistochemistry Staining
The procedure for immunohistochemistry was the same as what Li et al. reported[20]. Briefly, tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. Four µm thick paraffin sections were baked at 68°C for 90 min. Sections were then deparaffinized, rehydrated, antigen retrieval and quenched endogenous peroxidase. After blocking, rabbit anti-Ki-67 primary antibodies (1:200, Abcam, Cambridge, UK) were added and the slides were incubated at 4°C overnight. After washing three times, sections were incubated for 30 min with corresponding secondary antibodies at room temperature. Signals were detected with freshly made DAB substrate solution (ZSGB-BIO Company, Beijing, China).
Label-free Quantitative Proteomics (Label Free) Technology
Total protein of RPMI8226 cells was extracted followed by ultrasonic lysis and protein quantification. After trypsin digestion, liquid chromatography-mass spectrometry analysis was performed for peptide signal capture. Mass spectrometry data was retrieved through Maxquant (v1.6.5.0) supported by Uniprot Homo_sapiens database (Hangzhou Jingjie, China).
Immunoprecipitation-mass Spectrometry (Ip-ms)
RPMI8226 cells were collected and lysed for 30 minutes on ice. Soluble lysates were incubated with anti-PRAME (ab219650, Abcam) at 4°C overnight, followed by incubation of Protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) at 4°C for 2 hours. Immunocomplexes were eluted by Laemmli buffer and separated by SDS-PAGE. The separated gels were stained with Coomassie brilliant blue, and were cut into 6 molecular weight ranges and a heavy chain IgG band. Followed by stain removal and trypsin digestion of the cut gels, immunocomplexes were subjected to the Thermo Fisher Orbitrap Fusion Lumos Mass Spectrometer. IP-MS was assisted by Hangzhou Jingjie Biotechnology Co., Ltd (Hangzhou, China).
Immunoprecipitation And Endogenous Ubiquitination Assay
Cell extracts were incubated with primary antibodies PRAME (ab219650, Abcam) for co- immunoprecipitation (co-IP), p21 (ab109520, Abcam) and CTMP (14692-1-AP, Proteintech, Hubei, China) for endogenous ubiquitination assay or control IgG in a rotating incubator overnight at 4°C, followed by incubation with protein A/G Plus-Agarose (sc2003, Santa Cruz Biotechnology, TX, USA) for another 2 h. The immunoprecipitates were washed three times with lysis buffer and analyzed by immunoblotting with anti-CTMP and anti-p21 for co-IP assay, as well as anti-ubiquitin (sc8017, Santa Cruz Biotechnology, TX, USA) for ubiquitination testing.
Western Blot And Cycloheximide (Chx)-chase Assay
Cell lysates were prepared with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and analyzed by immunoblotting. Anti-PRAME (ab219650, Abcam), anti-p21 (ab109520, Abcam), anti-p27 (ab32034, Abcam), anti-CDK4 (23972, Cell Signaling Technology, MA, USA), anti-Bcl-2 (12789-1-AP, Proteintech), anti-Bax (50599-2-Ig, Proteintech), anti-PARP (9532, CST), anti-cleaved-PARP (5625, CST), anti-CTMP (14692-1-AP, Proteintech), anti-Akt (60203-2-Ig, Proteintech), anti-p-Akt (4060, CST), anti-CCND3 (66357-1-Ig, Proteintech) and anti-GAPDH (5174, CST) were used as primary antibodies.
For CHX-chase experiments, cells were treated with 50 µg/mL CHX (HY-12320, MedChemExpress, New Jersey, USA) in combination with 10 µmol/L MG-132 (HY-13259, MCE) or DMSO for 0, 2 h, 4 h and 8 h.
Statistical analysis
All data were presented as the mean ± standard deviation (SD). The Student’s t-test was used to determine significant differences between two groups, and one-way ANOVA was used to estimate differences between three or more groups. All P-values were obtained using two-tailed tests, and P < 0.05 was considered statistically significant. The statistical analysis was performed using SPSS software version 26.0 (SPSS, Inc., Chicago, IL) and GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA).