As previously described (He et al., 2020), the Fam3a promoter region (–3000 to 0 bp) was PCR-amplified and cloned into the pmirGLO of Dual Luciferase Expression Vector (Promega, Madison, WI, USA) to construct wild-type and mutated luciferase reporter vectors (Generay Biotech Co., Shanghai, China). Then, HEK293T cells (Chinese Academy of Sciences, Shanghai, China) were co-transfected with the reporter vectors and a plasmid encoding full-length mouse Hoxa5 or empty vector (GeneChem) using Lipofectamine 3000. The relative luciferase activities were measured 48 hours after co-transfection using a luciferase reporter gene assay kit (YPH, Beijing, China), following the manufacturer’s protocols.
Empty vector
Manufactured by Genechem
Sourced in China
An empty vector is a circular DNA molecule that serves as a backbone for cloning genetic material. It provides a basic structure for inserting and propagating DNA sequences of interest in host cells, such as bacteria or yeast. The empty vector itself does not contain any specific genetic information and is used as a tool for recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lentiviral vector system, by Genechem (2 mentions)
X tremegene sirna transfection reagent, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Scr sirna, by RiboBio (1 mentions)
Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions)
E2920, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Control vector, by Genechem (1 mentions)
11 protocols using empty vector
1
Fam3a Promoter Regulation by Hoxa5
2
Overexpression and Knockdown of TSPAN7 in Cells
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TSPAN7 and empty vector were purchased from Genechem (GENECHEM, Shanghai, China). TSPAN7-shRNA and negative controls were bought from Genechem (GENECHEM). Cells were transfected with plasmids using lipofectamine 2000 reagent (Gibco) and siRNA using X-tremeGENE™ siRNA Transfection Reagent (Sigma-Aldrich Co., St Louis, MO, USA) according to respective manufacturer’s instructions. The transfected efficiency was assessed 48 hours later on protein and mRNA level. All the following experiments after transfection/inference were performed at least in triplicate.
3
Overexpression and Silencing of ENTPD5 in Cancer Cells
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The lentiviruses for ENTPD5 overexpression, ENTPD5 shRNA and empty vector were purchased from Genechem (Shanghai, China). SKOV3 and OCAVAR8 cells in logarithmic growth phase were plated in 6-well plates with a planting density of 1 × 105 / well, and cultured in a 37 °C incubator. According to the instructions, when the cell growth density reached 30%, the infection was performed for 24 h. The puromycin medium was changed to screen positive cells at 24 h. The cells infected with ENTPD5 shRNA and overexpressed lentiviruses were recorded as sh-ENTPD5 group and LV-ENTPD5 group, and the cells infected with negative control were recorded as NC group. The sequences of short hairpin RNA (shRNA) targeting ENTPD5 were: 5′-CCAACACCATGCGTGTTGT-3′; The invalid shRNA sequences were: 5′-TTCTCCGAACGTGTCACGT-3′.
4
Overexpressing c-Myc in NSCLC cells
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Human NSCLC cell lines A549 and H460 (Shanghai Cell Bank, Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 medium with 10% FBS. The culture environment was 37°C and 5% CO2.
Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) was used to perform transient transfection according to the manufacturer's instructions. Human c-Myc expression vector and empty vector (catalog number: POSE146072729, GeneChem, Shanghai, China) were used for c-Myc overexpression and negative control, respectively.
Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) was used to perform transient transfection according to the manufacturer's instructions. Human c-Myc expression vector and empty vector (catalog number: POSE146072729, GeneChem, Shanghai, China) were used for c-Myc overexpression and negative control, respectively.
5
Overexpression of GPX4 in HT22 Cells
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HT22 cells in the GPX4 overexpression (GPX4-OE) group were transfected with Ubi-GPX4-CBh-gcGFP-IRES-Puro lentiviral vectors (GeneChem, Shanghai, China). Meanwhile, HT22 cells in the negative control (NC) group were transfected with the empty vector (GeneChem, Shanghai, China). For the stable overexpression of the GPX4 gene, the HT22 cells were seeded in 6-well plates and cultured for 24 h. The cells were then transfected with lentiviruses for 10 h and selected in 1.5 µg/mL puromycin (REVG1001, GeneChem, Shanghai, China). Moreover, the stably transfected cells were selected using 0.25 µg/mL puromycin. At last, the efficiency of overexpression was evaluated by Western blot analysis.
6
Glioma Cell Lines and Transfection Protocols
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These four glioma cell lines (A172, T98, U87 and U251) and a normal human astrocyte (NHA) cell line, which were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Chinese Academy of Medical Science, Shanghai, China), were maintained in our lab and cultured as previously described.15 (link) The miR-769-5p inhibitors (anti-miR-769-5p) and negative control inhibitors (NC) were obtained from RiboBio (Guangzhou, China). Lentiviral vector-mediated miR-769-5p inhibitors were purchased from GeneCopoeia (Guangzhou, China). pcDNA3.1-lysine methyltransferase 2A (KMT2A) and empty vector (EV) were obtained from Genechem (Shanghai, China). KMT2A siRNA (siKMT2A; 5′-GGT GTT GTC GTC GTT GCA AAT-3′) and corresponding scrambled control siRNA (Scr siRNA; 5′-TTC TCC GAA CGT GTC ACG T-3′) were synthesized by RiboBio. These plasmids and oligos were transfected into glioma cells using lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
7
Anaplastic Thyroid Carcinoma Cell Lines
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The human ATC cell lines 8305c (Cat NO. SCSP540) and FRO (Cat NO. SCSP577) were obtained from Cell bank of typical culture preservation Committee of Chinese Academy of Sciences. 8305c cell line was originally derived from anaplastic thyroid carcinoma of an adult female. FRO cell line was also originally derived from anaplastic thyroid carcinoma of an adult female. These cell lines have been authenticated by using Single Tandem Repeat (STR) profiling method. There is no mycoplasma contamination in 8305c and FRO cell lines. 8305c cells were cultured within MEM (the medium of the modified Eagle) supplemented in a humidified 5% CO2 incubator with 10% fetal bovine serum (FBS) at room temperature. FRO cells were cultured within DMEM (the medium of the modified Eagle) with 10% fetal bovine serum (FBS). MiR-155 ASO, mimic and negative controls were bought from Genecopoeia. Empty vector (EV) as well as SOCS1-specific siRNA and overexpression vector were gained from Shanghai Genechem Co., LTD. Cells were transfected by adopting the Lipofectamine 2000 kit (Invitrogen) in accordance with the instructions of the manufacturer.
8
Investigating HOXA-AS2 and Notch1 in Endothelial Cells
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Using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer’s instructions, HOXA-AS2 siRNA and respective negative control (NC) siRNA (RiboBio Corporation, Guangzhou, China), Notch1 overexpression plasmid vectors and empty vectors (GeneChem, Shanghai, China) were transfected into endothelial cells. For HOXA-AS2 overexpression lentivectors (GeneChem, Shanghai, China) infection, firstly we co-cultured the lentivectors with different cell density in different medium with or without Polybrene or ENi.S.(Enhanced Infection Solution) to find the proper multiplicity of infection (MOI) for lentivectors. Then, the selected condition, including cell seeding density and the most effective conditioned medium, was used in the subsequent infection experiments. The representative images of cell transduction efficiency by lentivectors were shown in Supplementary Material: Figure S1
9
Lentiviral Vector-Mediated Gene Silencing
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We ordered the lentiviral vector system, shRNAs, and empty vectors from GeneChem (Shanghai, China). Oligonucleotides for mimics, inhibitors, and negative control were obtained from RiboBio (Guangzhou, China). The miR-665 mimics and inhibitor were purchased from the same company. After culturing for 1 day, cells were transfected with plasmids. After 2 days, the cells were harvested, and their RNA was extracted.
10
Ntrk2 Promoter Regulation by Nr4a1
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HEK293T cells were incubated with the Nr4a1 (mouse)/NR4A1 (human)‐overexpressing plasmid or empty vectors (GeneChem, China) for 24 hours, while N2a cells were incubated with the Nr4a1‐knockdown shRNA plasmid and control vectors (GeneChem). The Ntrk2 (mouse)/NTRK2 (human) promoter and Nr4a1‐binding region of the truncated Ntrk2 promoter were cloned into the pGL3‐promoter vector with a luciferase reporter gene (Promega, USA). The luciferase reporter plasmid (1.0 µg) and TK‐Renilla (0.1 µg) were cotransfected into HEK293T cells or N2a cells in 24‐well plates using Lipofectamine 3000 (L3000015, Invitrogen). After 18 hours of incubation, firefly/Renilla luciferase activities were measured by a dual‐luciferase reporter system (E2920, Promega).
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