2.1 Regents. Reagents including antibodies, kits, chemicals (with work concentration) were all stated in suppl. table 2.
2.2 Cell culture. K562, Ba/F3-B/A and Ba/F3 cells were cultured in RPMI-1604 medium (HyClone, South Logan, UT, USA) supplemented with 10% of FBS (Biological Industries, Kibbutz Beit Haemek, Israel) (V/V). For Ba/F3 cells, IL-3 (PeproTech, Rocky Hill, NJ, USA) at 70 ng/ml was added. HEK-293 cells were cultured in DMEM medium (HyClone, South Logan, UT, USA) supplemented with 10% of FBS (Biological Industries, Kibbutz Beit Haemek, Israel) (V/V). All cells were maintained in 37 ℃, 5% CO2.
2.3 Isolation of PB-MNCs. Peripheral blood mononuclear cells (PB-MNCs) were isolated using human bone marrow mononuclear cell isolation kit (Tbd science, Tianjin, China) according to users` guide. Sample information was listed in Table S1. Informed consent forms were signed by all patients and healthy donors to allow the use of their cells for experiments and the experiments were performed in accordance with the regulation of institutional ethics committee of the First affiliated hospital of Xi’an Jiao tong university. Isolated cells were seeded in StemProTM-34 SFM (Gibco, Grand island, NY, USA), and used for following experiments immediately[8].
2.4 CCK-8 assay. Cells were seeded in 96-well plate (in the abovementioned mediums), etoposide (8 µM) was then added to make the final volume to 200 µL, for the duration of indicated days, 20 µL of enhanced CCK-8 was added, and the plate was then incubated in 37 ℃ for half hour. OD450 was then measured using microplate readers (BioTek, Biotek Winooski, Vermont, USA).
2.5 FoxM1 knockdown. hFOXM1 shRNA lentiviral was a product of Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were plated in 6-well plate (5⋅105/ml) in complete medium with 5 µg/ml of polybrene (Santa Cruz, CA, USA), after 30 min of incubation in 37 ℃, 4 µL of the stock solution of the FoxM1 shRNA lentiviral or scrambled control was added. The plate was then centrifuged at 150⋅g at room temperature for 4 hours, followed by 48 hours incubation in 37 ℃, 5% CO2. cells were collected for cycle and apoptosis analysis or protein extraction.
2.6 Construction of FoxM1 plasmid. Full-length FoxM1 was cloned from heathy white blood cells, double digested with EcoRⅠ and XhoⅠ TaKaRa (Tokyo, Japan), and cloned into pCMV-N-Flag backbone vector using T4 ligase TaKaRa (Tokyo, Japan). The ligation product was then transformed into competent DH5α cells and selected by colony PCR and double digestion, followed by sequencing.
2.7 Construction of CHK1 plasmid. Wide type CHK1 plasmid was constructed as FoxM1. The mutants were built by overlay PCR using the wide type as template. The primers were listed in suppl. materials. After obtaining the mutant DNAs, the followed steps (double digestion, ligation, transforming and selection) were as described in 2.6 section. Transfection was conducted using Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) according users’ manual. For each well of 6-well plate containing 5⋅105/ml cell, 2 ng of plasmid was used.
2.8 Western blotting. Cells post indicated treatments were harvested, washed three times with pre-cold PBS, lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Beyotime, Hangzhou, China. 1:50 v/v) on ice for 15 minutes, centrifuged at 12000 rpm at 4℃ for 5 minutes. The supernatants were collected for protein quantification by BCA (Beyotime, Hangzhou, China). SDS-PAGE was performed to separate the protein, briefly, 50 ng of protein for each well was loaded, electrophoresis using 10% of gel was ran for 2 hours at 120 V. The proteins were then transferred to PVDF membrane at constant current of 250 mA. Then the membrane was blocked in 5% of non-fat milk for 1 hour in room temperature and incubated with indicating antibodies (see suppl. table 2) at 4℃ overnight, washed in TBST buffer for three times, followed by secondary antibody incubation (1 hour, RT). The signals were detected using Odyssey® CLx Imaging System (LI-COR biosciences, Nebraska, USA).
2.9 Co-Immunoprecipitation. Co-Immunoprecipitation was performed according to the users' guide. Briefly, cells were lysed, and the lysate was pre-cleaned using control agarose resin followed by incubation with CHK1 or Flag antibodies to form immune complex which was then captured by protein A/G plus agarose, and centrifuged to separate the uncombined FoxM1. The pellets were washed and re-suspended with non-reducing lane marker sample buffer, boiled and centrifuged to collect elution for western blotting analysis.
2.10 Statistical analysis. The data were presented as X ± SD (n = 3). The difference between each group was evaluated using Student’s t-test. P < 0.05 was considered statistically significant.