DNA extraction and whole exome sequencing
Extract the whole genome DNA of blood according to the manufacturer’s instructions of TIANamp Genomic DNA Kit. Applied to Berrygenomics Bioinformatics Institute (Beijing, China) to provided whole exome sequencing services. Exomes were captured by Agilent SureSelect Human All Exon V6 kits were used to trapping exomes, Novaseq 6000 system was used for high throughput sequencing and the mutation validation confirmed by Sanger sequencing. The necessary bioinformatic analysis was also performed by the Berrygenomics Bioinformatics Institute.
Cell culture and transfection
MDA-MB-231 and MDA-MB-468 were cultured in DMEM containing 10% fetal bovine serum at 37℃ and 5% CO2. The cell culture medium was changed every 2 days. According to the manufacturer's instruction, 5-10μL Hieff TransTMLiposomal Transfection Reagent (Cat# 40802ES03, Yeasen,China )and 2-5μg plasmid were used to transfect MDA-MB-231 and MDA-MB-468. QPCR and Western blot confirmed its transfection efficiency.
Plasmid and stable cell line construction
3×flag-RBBP8-WT and 3×flag-RBBP8-841 were cloned into CD513B vector. RBBP8 cloning primer: RBBP8-WT/841-F: CGGGATCCATGAACATCTCGG- GAAGCAGCT, RBBP8-WT-R: ccGCGGCCGCTGTCTTCTGCTCCTTGCCT, RBBP8-841-R: cgGCGGCCGCCTAATCACCAAGGGGGCTCATGGGA; The constructed plasmid was transfected into 293T cells with helper plasmids (pMD2.G and psPAX2) by Calcium transfer method. The lenti -virus supernatant was collected every 24h for 3 days. All virus solutions were concentrated with 20% sucrose, 2-5×106 cells were mixed with 1 ml of virus solution and 10 μg/ mL Polybrene (Cat# 40804ES76, Yeasen,China) was incubated for 8h at 37 ℃. Then change the solution with 2 mL of fresh complete medium, continue to culture for at least 48h, added 2ug /mL puromycin for 3 days to obtain stable over- expression cell line. The effect of over-expression was detected by QPCR and Western Blot.
CCK8
Cells was digested with trypsin, and resuspended with 4ml complete medium, then adjust the cell density to 2×104/mL, 100μL cell suspension inoculated into 96 well plates. 24h, 48h, 72h, 96h later, 10μL CCK-8(Cat# C0005, Target Mol, USA) was added into 96 well plates , incubation at 37 ℃ for 2h-4h. The OD value was measured at 450 nm by microplate assay(EnSpire 2300, PerkinElmer, USA). Repeat each group three times.
Clone formation assay
Cells was digested with trypsin, and resuspended with 4ml complete medium, then dilute the density of cell to 10×104/mL, 100μL cell suspension inoculated into 6 well plates. 2mL complete medium was added into 6-well plate, Change the complete medium every 2 days. 7-10 days later, discard the culture medium and wash it with PBS for three times, stain with 0.1% crystal violet(Cat#G1063, Solarbio, China) solution for 30mins, wash with PBS for three times, then take photos under the microscope and calculate the number of clones.
Transwell
Cells was digested with trypsin, and resuspended with 4ml complete medium, then dilute the density of cell to 10×104/mL. 200μL cell suspension were inoculated into the upper chamber without FBS and 750μL medium with 10% FBS was added into the lower chamber. 48h later, discard the medium, wash with PBS for three times, stain with 0.1% crystal violet, washed, dried and counted.
Western Blot(WB)
The cells were washed three times with PBS and lysated with RIPA+1% Phosphorylase inhibitors and Protease inhibitors on ice for 30mins, then collect supernatant with 12 000rpm, 10mins. Protein quantification by Bradford method (Cat#PC0010, Solarbio, China). The same amount of protein was separated with 10% SDS-PAGE Gel, then 0.45um PVDF membrane(Cat# IPVH00010, Millipore) was transferred membrane with 250mA for 2h, Blocked with 5% no fat milk for 1h. Then incubate the primary antibody of E-cadherin(1:1000, Cat#14472, Cell Signaling Technology, USA), GAPDH(1:1000, Cat#AP0063, Bioworlde, USA) overnight, wash with TBST twice for 8mins, incubate the secondary antibody(1:5000, Cat#AP307P, sigma, USA)for 2h, then wash with TBST twice for 8mins, and blotted with chemiluminescence detection system (BIO-RAD, USA).
Immunofluorescence
Cells were immobilized with 4% paraformaldehyde for 20 min, PBS washed three times. 0.2% TritonX-100/PBS, PBS permeating cells 10 mins, PBS washed three times. then 5% BSA/PBST blocked for 2h, incubate the primary antibody of HA(1:1000, Cat#3724, Cell Signaling Technology, USA) overnight, wash with PBST twice for 8mins. incubate the goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor 594(1:1000, Cat#AWS0006b, abiowell, China) for 2h, then wash with PBST twice for 8mins, later, 1ug/mL DAPI(Cat#FXP139-100, 4A biotech, China) staining cell nucleus for 5 mins, wash with PBST three times for 8 minutes. Image acquisition under fluorescence microscope( AXIO VERT.A1, ZEISS, Germany).
Nuclear and cytoplasmic protein extraction
Nuclear protein and cytoplasmic protein were isolated by nuclear and cytoplasmic protein extraction kit( Cat#P0027, beyotime, China). Then protein was separated with 10% SDS-PAGE Gel, then 0.45um PVDF membrane(Cat# IPVH00010, Millipore) was transferred membrane with 250mA for 2h, Blocked with 5% no fat milk for 1h. Then incubate the primary antibody of Tubulin(1:1000, Cat#TA373086, Origene, China), Histone H3(1:1000, Cat#4499, Cell Signaling Technology, USA), HA(1:1000, Cat#3724, Cell Signaling Technology, USA) overnight, wash with TBST twice for 8mins, incubate the secondary antibody(1:5000, Cat#AP307P, Sigma, USA)for 2h, then wash with TBST twice for 8mins, and blotted with chemiluminescence detection system (BIO-RAD, USA).
Immunocoprecipitation(CoIP)
The cells were washed three times with PBS and lysated with IP cell lysate+1% Phosphorylase inhibitors and Protease inhibitors on ice for 30mins, then collect supernatant with 12 000rpm, 10mins. Added 20uL Anti Flag immune magnetic beads, overnight at 4 ℃. 500uL TBST cleans magnetic beads 5 times for 5mins. Protein separation, membrane transfer and detection according to WB experimental steps. The primary antibody of FLAG(1:1000, Cat#F1804, Sigma, USA), BRCA1(1:1000, Cat#14823, Cell Signaling Technology, USA).
In vivo xenograft
MDA-MB-468 cell density of 2×107/mL, 150μL cell suspension combined with 150ul Matrigel medium(Cat#356234,Corning, USA) were injected into nude mice. Observe and measure the volume of tumor every week, 4 weeks later, the mice were executed by cervical vertebra dislocation. Repeat each group 5 times.
Statistical analysis
Data calculations were performed using Prism 8.0 (GraphPad, San Diego, CA), three independent data are presented as means ± SD. Two-tailed Student’s t-test or ANOVA one way was used for analyses compared two groups or multiple groups. P<0.05 was considered statistically significant (*P< 0.05, **P< 0.01, ***P< 0.001 represents compared with control group.)