Chitosan-oligosaccharides (CSO, 970Da) were obtained from cool chemical science and technology (Beijing, China), and the degree of deacetylation of CSO is 95%. Cystamine dihydrochloride (98%) and acryloyl chloride (97%) were purchased from Bailingwei Technology Company (Beijing, China). Jiangsu Qiangsheng Functional Chemical Company provided dichloromethane (DCM; analytical grade). Lipo8000™ was provided by Beyotime Biotechnology (Shanghai, China). Fetal bovine serum (FBS), trypsin, and penicillin-streptomycin were purchased from Gibco (USA). Dulbecco’s modified eagle’s medium (DMEM) and Lipo8000™ were supplied by Beyotime Biotechnology (Shanghai, China). GFP antibody, CAT plasmid with N terminal GFP Spark tag, and GFP plasmid was obtained from Sino Biological (Beijing, China). The Fourier-transform infrared spectrometer (FT-IR) is from Thermo Fisher Scientific’s Nicolet™ iS50 FTIR Spectrometer series (Massachusetts, USA). The solid microneedles are from Shanghai Fanzhen Trading Co., Ltd. Catalase detection kits, and Western and IP cell lysates, fixatives, and blocking solutions were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Nanjing Jiancheng Bioengineering Institute provided alanine aminotransferase and aspartate aminotransferase detection kits (Nanjing, China).
Lipo8000
Manufactured by Beyotime
Sourced in China, United States
The Lipo8000 is a high-performance lithium-ion battery testing system. It is designed to accurately measure and analyze the performance characteristics of lithium-ion batteries. The Lipo8000 provides real-time data collection and analysis capabilities to support battery research and development efforts.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Dual luciferase reporter assay system, by Promega (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Polybrene, by Beyotime (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
X tremegene sirna transfection reagent, by Roche (2 mentions)
Microplate spectrophotometer, by Bio-Rad (2 mentions)
Luciferase reporter gene assay kit, by Beyotime (2 mentions)
Pgl3 basic vector, by Promega (2 mentions)
Dual luciferase assay kit, by Vazyme (2 mentions)
Lipornai, by Beyotime (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Polybrene, by Solarbio (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
123 protocols using lipo8000
1
Chitosan-oligosaccharide-based Transdermal Delivery
2
Establishing Stable circIFNGR2-Expressing THP-1 Cells
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Lentivirus was used for construction of circIFNGR2 stably-expression THP-1 cells previous described.6 (link) Briefly, the plasmids containing the sequences for overexpression or knockdown of circIFNGR2 were co-transfected with pSPAX2 and pMD2.G into 293T cells by Lipo8000 (Beyotime, Beijing, China). After collection and filtration, the supernatants were used for infecting THP-1 cells with 5 μg/mL polybrene (Beyotime, Beijing, China). Then, infected THP-1 cells were re-seeded in fresh medium and added with puromycin for 7 days. For miRNA and plasmids transfection, miR-939 mimics, miR-939 inhibitor, plvx vectors containing sequences of eIF4A3, si-eIF4A3 and controls were transfected by Lipo8000 (Beyotime, Beijing, China). qRT-PCR was used to evaluate the efficiency of infection and transfection.
3
Immortalized Chicken Preadipocyte Cell Line (ICP1) for Adipogenesis Studies
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The immortalized chicken preadipocyte cell line (ICP1) was obtained from the Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Northeast Agricultural University (Harbin, China). The ICP1 cell line was established by infecting chicken stromal-vascular cells with a recombinant retrovirus expressing chicken telomerase RNA
[11] (link); these cells share common morphology and differentiation features with primary preadipocytes and are widely used in chicken adipogenesis studies [
14 (link)–
16 (link)] . ICP1 and DF-1 cell lines (ATCC, Manassas, USA) were cultured in DMEM F-12 (Gibco, Carlsbad, USA) and DMEM high glucose (Gibco) respectively, supplemented with 10% fetal bovine serum (Gibco) and maintained at 37°C in a 5% CO
2 cell incubator [
11 (link),
17 (link)] . When cell confluence reached 60%‒70%, the cells were transfected using Lipo8000 (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
[11] (link); these cells share common morphology and differentiation features with primary preadipocytes and are widely used in chicken adipogenesis studies [
14 (link)–
16 (link)] . ICP1 and DF-1 cell lines (ATCC, Manassas, USA) were cultured in DMEM F-12 (Gibco, Carlsbad, USA) and DMEM high glucose (Gibco) respectively, supplemented with 10% fetal bovine serum (Gibco) and maintained at 37°C in a 5% CO
2 cell incubator [
11 (link),
17 (link)] . When cell confluence reached 60%‒70%, the cells were transfected using Lipo8000 (Beyotime, Shanghai, China) according to the manufacturer’s instructions.
4
Transfection of miRNA Mimics and Inhibitors
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The sequences of miRNA mimics (RiboBio, Guangzhou, China) and inhibitors (RiboBio) are listed in Table S1 . We confirmed that the miR-NCs, which were derived from cel-miR-293b-5p and cel-miR-67-3p, did not specifically target any mRNAs in the human genome. The cells were seeded into six-well plates at 60%–80% confluence. The next day, the medium was replaced with fresh medium containing miRNA mimics (150 μM) or inhibitors (200 μM) and Lipo8000 (Beyotime) as the transfection reagent. The cells were harvested for subsequent analyses 48 h after transfection.
5
Optimized Transfection Protocol for Gene Expression
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The experimental procedure was performed as follows: the cells were seeded into 6-well plates and allowed to reach 80% confluence. Transfection was performed using lipo8000 (Beyotime, Shanghai, China), and each well was transfected with a total of 2000 ng of plasmid DNA. This transfection protocol was selected based on preliminary experiments to determine the optimal transfection conditions for our specific cell line and plasmid constructs. The total RNA was extracted from the cells with a Simply P Total RNA Extraction Kit (Bioer Technology, Hangzhou, China) according to the manufacturer’s instructions. The RNA samples were immediately stored at −80 °C. Then, cDNA was made from 1000 ng of total RNA using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and they were used for qPCR.
6
Molecular Regulation of CROT Expression
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Small-silencing RNA (siRNA) of CROT and negative control, miR-33a-5p mimics, inhibitor, and negative control were synthesized by Shanghai Gene Pharma Co., Ltd (Shanghai, China). The sequences are listed in Supplementary Table S1
7
Overexpression and Knockdown of LINC01082 in GC Cells
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The human GC cell line HGC-27 was purchased from the Procell Life Science&Technology Co., Ltd (Wuhan, China). The cells were cultured in RPMI-1640 medium (#PM150110, Procell) with 20% fetal bovine serum (#164210-500, Procell) and 1% penicillin–streptomycin (#PB180120, Procell) in an environment with 5% CO2 at 37 °C. The cells were seeded in 6-well culture plates, and cell transfection was performed when the cell confluence reached 60–70%. For LINC01082 overexpression, the pcDNA3.1-LINC01082 plasmid was obtained from GENEWIZ (Suzhou, China). The negative control group of empty vector was referred to as the NC group. For LINC01082 knock down, the si-LINC01082 and the corresponding negative control were synthesized from Ribobio (Guangzhou, China). The cells were transfected with 2.5 µg plasmid DNA and 4 µl Lipo8000™ (Beyotime, China) per well following the manufacturer’s protocol. All cells were collected 48 h after transfection.
8
Lentivirus-Mediated CERS6 Knockdown
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Lentivirus was used for the construction of the CERS6 knockdown model in vitro and in vivo. We transfected pSPAX2 and pMD2.G with plasmids containing the sequences for knockdown of CERS6 or scrambled control into 293 T cells (20181122-01, Suyan, Guangzhou, China) using Lipo8000 (C0533, Beyotime, Beijing, China). After collection and filtration, the supernatants were used directly for cell infecting or concentrated by lentivirus concentration reagent (BF06205, Biodragon, Suzhou, China) for mouse experiments. Tsingke Biotechnology (Beijing, China) designed and produced the plasmids, and the shRNA sequence is presented in Table 1 .
9
Constructing circKRT7 Interference Plasmid
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In order to construct the circKRT7 interference plasmid, two synthetic hairpin nucleic acids were annealed and inserted into the pENTR/H1/TO vector. The primer sequences are as follows: Top Strand5ʹ-CACCGACCAAGGTACGAAGATGAAACGAATTTCATCTT CGTACCTTGGTC-3ʹ, Bottom Strand:5ʹ-AAAAGACCAAGGTACGAAGA TGAAATTCG TTTCATCTTCGTACCTTGGTC-3ʹ. The 322-base length circKRT7 nucleic acid sequence was synthesized and cloned into pLCDH-ciR vector and sequenced for identification. The antisense oligonucleotide sequence of miR-29a-3p was synthesized and used to block the level of miR-29a-3p. All plasmids and oligo were transfected with lipo8000 (Beyotime Biotechnology, China) into SKOV3 and ES-2 cells according to the manufactures.
10
Cell Culture and Compound Characterization
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Vero E6
and A549-ACE2 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin–streptomycin. In the case of Calu-3 cells, the medium
was also supplemented with 1% non-essential amino acids and 1% sodium
pyruvate. All cell lines were obtained from Otwo Biotech Corporation
and incubated at 37 °C with 5% CO2. Lipo8000 (Beyotime,
China) and pcDNA3.1-ACE2-Flag (Beyotime, China) were used for transfection.
The expression of ACE2 was tested by western blot (Figure S3 ). Chloroquine diphosphate salt was purchased from
Sigma-Aldrich (San Louis, MO, C6628; purity, 98.5–101.0%).
Hydroxychloroquine sulfate was purchased from Sigma-Aldrich (San Louis,
MO, 90527; purity, ≥95%). Favipiravir was purchased from Topscience
(China, T6833; purity, ≥98%). Remdesivir was purchased from
Cayman Chemical (Ann Arbor, MI, 30354; purity, ≥98%). GS-441524
was purchased from Selleck Chemicals (Houston, TX, S6814; purity,
≥99%). All chemicals were analyzed by HPLC (Figures S4–S8 ).
and A549-ACE2 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin–streptomycin. In the case of Calu-3 cells, the medium
was also supplemented with 1% non-essential amino acids and 1% sodium
pyruvate. All cell lines were obtained from Otwo Biotech Corporation
and incubated at 37 °C with 5% CO2. Lipo8000 (Beyotime,
China) and pcDNA3.1-ACE2-Flag (Beyotime, China) were used for transfection.
The expression of ACE2 was tested by western blot (
Sigma-Aldrich (San Louis, MO, C6628; purity, 98.5–101.0%).
Hydroxychloroquine sulfate was purchased from Sigma-Aldrich (San Louis,
MO, 90527; purity, ≥95%). Favipiravir was purchased from Topscience
(China, T6833; purity, ≥98%). Remdesivir was purchased from
Cayman Chemical (Ann Arbor, MI, 30354; purity, ≥98%). GS-441524
was purchased from Selleck Chemicals (Houston, TX, S6814; purity,
≥99%). All chemicals were analyzed by HPLC (
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