The PCDH-CMV-MCS-puro vector was employed to construct a full-length cDNA (GenBank accession number: NM_91607) expression vector for human SLFN11. The lentiviral supernatant was obtained after transfection into HEK293 cells by lipofectamine 3000 growing for 48 h and 72 h following the instructions of the manufacturer (Invitrogen, No. L3000008). Subsequently, the lentiviral supernatant was added into the culture medium. Puromycin (MCE, No. HY-15695) was then used to screen for SLFN11 expressing KYSE30 (2.5 μg/mL) and KYSE450 (1.0 μg/mL) cells 48 h after lentiviral transfection, with a duration of 3 d for the screening process. The monoclonal SLFN11 expression cells were obtained through limited dilution in 96 well plates, and further validated via Western blot.
L3000008
Manufactured by Thermo Fisher Scientific
The L3000008 is a laboratory instrument designed for performing spectral analysis. It is capable of measuring and analyzing the absorption or emission spectra of samples across a range of wavelengths. The core function of this product is to provide precise spectral data for research and analytical applications.
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7 protocols using l3000008
1
Overexpression of SLFN11 in Cancer Cells
2
Stable β-catenin Overexpression in HCT-116 Cells
3
Plasmid Transfection Using Lipofectamine
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Ub-G76V-GFP and Ub-R-GFP were purchased from addgene. Cells were transfected with plasmids using lipofectamine 3000 (invitrogen L3000008) according to the manufacturer’s protocol.
4
Exploring miRNA-mediated gene regulation
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At day 0 (D0), PDCs Ge904, chosen because they do not express miR-340 and miR-17-3p, were seeded at 80,000 cells per well in a 24-well plate. Twenty-four hours later at D1, cells were transfected with the corresponding miRNA and plasmid using Lipofectamine 3000 according to the manufacturer’s protocol (L3000-008 Invitrogen). Twenty-four hours later at D2, cells were lysed in the wells using the passive Lysis buffer from the Dual Luciferase Reporter Assay (E1910 Promega), followed by a measurement of the luminescence according to the manufacturer’s protocol.
Results were normalized to Renilla control.
Results were normalized to Renilla control.
5
Neuronal Regulation of Aβ Secretion
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2-wk-old differentiated cortical neurons were transfected with T7-RelA (S536E) plasmid (plasmid 24156; Addgene) using Lipofectamine 3000 and following the manufacturer’s instructions (L3000008; Thermo Fisher). Cells were treated with vehicle or 3K3A-APC (5 nM) for 24 h. Neuronal cells were collected for analysis of NFκB-p65 and BACE1 with immunoblotting, and relative abundance of RelA and Bace1 mRNA by real-time quantitative PCR (RT-qPCR). Neuronal culture medium was collected for analysis of human Aβ40, Aβ42, and Swedish sAPPβ levels with ELISA, as described below.
6
Efficient Generation of Transgenic Cell Lines
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All transgenic cell lines were made by transfecting cells at 30% confluence with 500 ng of appropriate HypG3 plasmid via 1 µL of lipofectamine 3000 (L3000008, ThermoFisher) per well of 24-well plate containing 500 µL of culture medium. The reagent was removed after 24 h, and fresh culture medium was added. Cells were allowed to recover for 24 h prior to selection with 1000 µg/mL G418 or 200 µg/mL hygromycin. After selection, cells were serially diluted into 96-well plates, and single colony clones were expanded. Clones showing robust growth and strong expression were chosen for subsequent experiments.
7
Knockdown of Circular RNAs in Prostate Cancer
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All small interference RNAs (siRNA) were synthesized by Hippo Biotechnology Company. DU145 and PC3 cells were seeded into the 6-well plates, then the negative control siRNA and siRNAs targeted to objective genes mixed with lipofectamine 3000 (L3000008, ThermoFisher) were added to the prepared cells in the fusion of 60–70%. RT-qPCR was used to detect the knockdown efficiency in DU145 and PC3 cells. Cell proliferation assay was carried out with CCK-8 kit (C0042, Beyotime Biotechnology),and cell viability was evaluated by the absorbance at 450nm using the microplate reader (Bio-Rad iMark). More details can be found in our previous study (Mo et al., 2017 (link)). SiRNAs sequence targeted to circ_14734 and circ_17720 are shown in Supplementary Table 1 .
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