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  • br For overexpression purposes human

    2020-08-12


    For overexpression purposes, human APPBP2, PPM1D, and SPOP CDS were cloned into pCDNA 3·1, which was delivered into the cell lines by lipo-3000 (Invitrogen, L3000008). The expression of the genes was driven under CMV promoter.
    For BrdU in A549 (or H1299): 5 × 105 sh-NC infected cells and sh-APPBP2 infected cells were cultured into 12-well plates. When the cells were attached to the wall, the investigators added BrdU to each well with a final concentration of 10 μM and cultured these for another 8 h. They then replaced the BrdU containing medium with normal me-dium (10% FBA in DMEM) and further cultured for 24 h. The cells were harvested and underwent immunostaining of BrdU (abcam, RRID: ab142567, 1:500) and DAPI.
    For MTT in A549 (or H1299), a modification of the method described by Mossman [10] was used in our experiments: The stable cells of sh-APPBP2 and sh-NC (transfected with plasmids or not) were cultured for 48 h. The investigators then transplanted the cells into 96-well at the appropriate seeding density of 5000 cells/well. Cells were incubated with 10 μl MTT (5 mg/ml) for 3 h, then the medium was replaced with 150 μl 100% dimethylsulfoxide (DMSO) in each well. Plates were then agitated on a plate shaker for 5 min, following which, spectrophotomet-ric absorbance at 570 nm was immediately determined using a scanner.
    2.7. Colony formation assay
    A549 (or H1299) cells were treated with sh-APPBP2 or sh-NC lenti-virus (10 x cell number/viral titer) and cultured for 48 h. The cells were then disassociated and seeded for colony formation in 6-well plates at two hundred viable cells (the density was measured by trypan blue assay) per well. After 14–21 days (medium exchanged every three days), colonies were scored using a microscope by adding crystal violet staining at a concentration of 0·4%. Colonies were counted only if a sin-gle clone contained N100 cells. Each assay was performed in triplicate on two independent occasions. For functional rescue experiments, after cells were transfected with PPM1D or SPOP plasmids for 48 h (Fig. 5a), 500 viable cells were seeded for colony formation in 6-well plates. 
    2.8. Cell sorting for apoptosis and T16AinhA01 assay
    To study the impact of APPBP2 on apoptosis, 106 stable cells of A549 (or H1299) were stained with Annexin V-APC and 7-AAD (Biolegend, 640930, USA) according to the manufacturer's instructions. The apopto-tic cell proportion was assessed with flow cytometry (BD, Calibur, USA). To study the impact of APPBP2 on the cell cycle, cells (4 × 105) were fixed in 70% ethanol for 1 h at 4 °C. Then the cells were washed twice with PBS followed by adding 10 mg/ml RNase A. The investigators then added propidium iodide (BD, 550825) at a final concentration of 0·05 mg/ml. The samples were incubated at 4 °C for 30 min in a dark en-vironment. Finally, samples were analysed by flow cytometry. All the assays were performed in triplicate. To study the rescue function of PPM1D or SPOP on apoptosis, A549 (or H1299) stable cell lines were transfected with 1 μg PPM1D or SPOP in a 6-well plate to culture for 48-h, and then underwent apoptotic assessment.
    2.9. Wound healing assay
    Wound healing assay was performed according to a previously de-scribed method with some modifications [11]. A549 (or H1299) stable cells were transplanted into 6-well plates at the appropriate seeding density of 1 × 106 cells/well. While confluent, the cell monolayer was mechanically scarred with a sterile 200 μl pipette tip and incubation was continued for an additional 12 h and 24 h. Images were taken at 0, 12, and 24 h after scarring.
    In vitro invasion capability was measured using 24-well chambers (Millipore, 8 um, USA) pre-coated with Matrigel (BD, 256234) on the polyethylene terephthalate membrane. In the upper compartment of the transwell chambers, the investigators seeded 5 × 104 stable cells suspended in 500 μl of serum-free medium and filled the lower com-partment with complete medium. After incubation at 37 °C under 5% CO2 for 6 to 10 h, some of the cells had migrated through the porous membrane, whereas the non-migrating cells remained on the upper surface of the filter. The chamber was then immersed in methanol for 10 min to fix the cells, and then scrubbed gently to remove the non-metastatic cells. The migrated cells were stained with 0·5% crystal violet for 10 min and counted under a light microscope. Each experiment was independently performed three times. For Fig. 6a,b, the A549 (or H1299) stable cells were further transfected with PPM1D or SPOP plas-mid by lipo-3000. The cells were cultured for 48 h and then examined.
    2.11. Establishment of an adenocarcinoma xenograft mouse model