To construct the CIAV VP1 and VP2 co-expressing plasmid, VP1 and VP2 fragments were amplified through PCR using the primers listed in Table 1 . Then, the purified PCR product of VP2 was ligated to the pBud CE 4.1 vector (Invitrogen, Carlsbad, CA, USA) and digested by restriction enzymes BamH I and Sal I to construct plasmid pBud-VP2. After that, the purified PCR product of VP1 was ligated to pBud-VP2 and digested by restriction enzymes Kpn I and Xho I to construct recombinant plasmid pBud-VP1-VP2. To verify the successful expression of pBud-VP1-VP2 in eukaryotic cells, 2 × 105 DF-1 cells were inoculated in two 24-well culture plates. Then, the pBud-VP1-VP2 plasmid was transfected into DF-1 cells using liposome 2000 (Thermo Fisher, Waltham, MA, USA) according to the instruction manual. After transfection, the cells were cultured in a 5% CO2 incubator at 37 ℃ for another 10 h and changed to complete DMEM medium containing 10% fetal bovine serum (FBS). The protein expression was verified by Western blot analysis and IFA assay.
Liposome 2000
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
Liposome 2000 is a laboratory equipment designed for the formation of liposomes, which are spherical vesicles composed of lipid bilayers. It provides a controlled and reproducible method for the preparation of liposomes, which are widely used in various applications such as drug delivery, gene transfection, and membrane studies.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Pbudce4.1 vector, by Thermo Fisher Scientific (1 mentions)
Stat3 sirna, by RiboBio (1 mentions)
Mir 29a mimic, by RiboBio (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Glomax 20 20 luminometer, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Stat3 sirna, by GenePharma (1 mentions)
Mir 125b 5p mimic, by GenePharma (1 mentions)
Pcdna3.1 stat3, by GenePharma (1 mentions)
Mir 125b 5p inhibitor, by GenePharma (1 mentions)
Prl tk vector, by Promega (1 mentions)
Mir 149 mimic, by GenePharma (1 mentions)
Pore size filter, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Shrna nc, by GenePharma (1 mentions)
Mir nc, by GenePharma (1 mentions)
Mir 181b mimic, by GenePharma (1 mentions)
32 protocols using liposome 2000
1
CIAV VP1 and VP2 Co-Expression Plasmid Construction
2
miR-29a and STAT3 Regulation in EC Cells
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Human EC cell line HHUC was purchased from the Cell Resource Center of Shanghai Institute of Biosciences, Chinese Academy of Sciences. EC cells were stored in DMEM with 10% fetal bovine serum at 37 ℃ and in 5% CO 2 . miR-29a mimics or inhibitors, STAT3 plasmids (overexpressed STAT3 and STAT3-OE) or shRNA (knockdown of STAT3 and STAT3-KD) were transfected into 6-well plates. Liposome 2000 (Thermo Fisher Science) with untreated HHUC were used as the negative control (NC). miR-29a mimics, miR-29a inhibitors, STAT3 plasmids (pcDNA3.1-STAT3) and STAT3-siRNA were all purchased from Ribobio (RiboBio Co.Ltd., China).
3
Overexpression and Knockdown of PTBP1 in Gastric Cancer
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The siRNAs were designed and produced by Gene Pharma (Additional file: Table S1 ). Briefly, cells seeded overnight in 6-well plates were transfected with siRNA using Liposome 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and incubated for 6 h before replacement with a serum-containing medium. Transient overexpression of PTBP1 was performed by inserting the CDS region of PTBP1 into the pcDNA3.1 vector. Full-length and truncated PTBP1 plasmids were constructed in the pCDH vector. Neofect (Neofect, Beijing, China) was used to transfect plasmids into GC cells. To construct cell lines with a stable knockdown of PTBP1, we used the HBLV-LUC-PURO vector and synthesized the lentivirus at HanBio (Shanghai, China). The lentivirus sequence was identical to that of siRNA (Additional file: Table S1 ). HGC27 cells (Chongqing, China) were seeded equally into 24-well plates and placed in an incubator overnight. Lentiviral vectors encoding small interfering RNA targeting PTBP1 and control lentivirus CON207 were then transfected. Next, they were treated with puromycin (2 μg/mL) for 1 week. The transfection efficiency of the virus was verified via Western blot, and these cells were used for subsequent experiments.
4
Generating Stable hAAT-Expressing NIT-1 Cells
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NIT-1 cells (a kind gift from Prof. Li Fangping, Sun Yat-Sen University, China), an insulin-producing insulinoma cell line, derived from non-obese diabetic (NOD) mice prone to autoimmune diabetes [14] (link) were used as a cell model system. These cells were expanded in 24-well tissue culture plates in Dulbecco's modified Eagle's medium (DMEM; Sigma, St. Louis, MO, USA) with 10% FCS (Gibco, CA, USA). Liposome 2000 (Invitrogen, Carlsbad, CA, USA) was utilized to transfect pDsRed–hAAT or pDsRed mock-vector into the NIT-1 cells respectively. Seventy-two hours after the transfection, G418 (350 µg/mL, Sigma) was added to the medium for selection. Low-dose G418 (175 µg/mL) was subsequently applied to generate cells stably expressing the construct. The 10th and the 40th generation of the NIT-hAAT cell line were collected and lysed. Western blotting was conducted to validate that hAAT was expressed at the protein level. The hAAT levels from cultured supernatant samples (72 h) at passage 10 and passage 40 (5×105 cells/mL)were determined by Human Alpha 1-Antitrypsin ELISA(Immunology Consultants Laboratory, Newberg,OR, USA).
5
Modulating miR-361-3p Expression in Cells
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As part of this study, cell transfer was carried out to change the expression level of miR-361-3p. The miR-361-3p mimic and miR-361-3p inhibitor were designed and synthesized by a gene pharmaceutical company in Shanghai, China.
The miRNA mimic sequence is as follows: miR‐361-3p mimic: 5ʹ-UCCCCCAGGUGUGAUUCUGAUUU-3ʹ, miR‐361-3p inhibitor: 5ʹ-AAAUCAGAAUCACACCUGGGGGA-3ʹ.
Cells were inoculated into 6-well plates 24 hours prior to transfection. When the cell fusion reached 60–70%, the cells were transfected with liposome 2000 (Invitrogen) quickly. After staining for 6 hours, the medium was changed, and after being cultured for 48 hours, the cells were collected for subsequent experiments.
The miRNA mimic sequence is as follows: miR‐361-3p mimic: 5ʹ-UCCCCCAGGUGUGAUUCUGAUUU-3ʹ, miR‐361-3p inhibitor: 5ʹ-AAAUCAGAAUCACACCUGGGGGA-3ʹ.
Cells were inoculated into 6-well plates 24 hours prior to transfection. When the cell fusion reached 60–70%, the cells were transfected with liposome 2000 (Invitrogen) quickly. After staining for 6 hours, the medium was changed, and after being cultured for 48 hours, the cells were collected for subsequent experiments.
6
Dual Luciferase Assay for Polβ Promoter Analysis
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We resuspended 293T cells (2 × 105 cells per well on 24‐well plate) which were subsequently transfected with 600 ng pGL4‐polb plasmid and 50 ng pRL‐TK plasmid with liposome 2000 (Invitrogen), following the manufacturer's recommended protocol. Six hours after transfection, cells were cultured with 500 μl fresh DMEM medium containing 10% FBS for additional 48 h. Cell lysates were then prepared with 200 μl passive lysis buffer and 20 μl were used for the Firefly luciferase (Fluc signal) and Renilla luciferase (Rluc signal) activity measurement by dual luciferase reporter assay system on GloMax 20/20 luminometer using the standard detection parameter (Promega). The transcriptional activity of each promoter was calculated with Fluc/Rluc ratio. The ratio of wild‐type polb gene promoter was set to 1 and ratios of other mutated polb promoters were relative to that of wild‐type and expressed with x ¯
7
THP-1 Cell Transfection with miR-433-3p Inhibitor
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THP-1 cells were treated with P-sEVs or LPS pre-sEVs, respectively, on a 6-well culture plate, and then were transfected with miR-433-3p inhibitor or negative control at the final concentration of 50 nM using liposome 2000 (Invitrogen). The cells were harvested 48 h after transfection and used in the following experiment.
8
Overexpression of TAB3 in Nucleus Pulposus Cells
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Firstly, the open reading frame (ORF) of the TAB3 gene was amplified with cDNA of NP cells as a template, and then connected with linearized pcDNA3 to construct the recombinant plasmid pcDNA3-TAB3. Following that, the pcDNA3-TAB3 was amplified. The amplified fragment was combined with the eukaryotic expression vector pEGFP to construct the recombinant plasmid pEGFP-TAB3 (Clontech Laboratories, Inc., Mountain View, CA, USA). MiR-16 mimics and the miR-16 inhibitor were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). NP cells were transfected with miR-16 mimics, the miR-16 inhibitor and TAB3 over-expressing plasmids. The co-transfection was conducted in NP cells with miR-16 mimics and TAB3 over-expressing plasmids. Liposome 2000 (Invitrogen Inc., Carlsbad, CA, USA) was used for transient transfection of NP cells. After cell transfection, NP cells were stimulated with LPS (10 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) for various lengths of time.
9
Targeted silencing of hsa_circ_0005991 in CAL27 cells
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We employed a negative control (NC) oligonucleotide and short interfering RNA (siRNA) to target hsa_circ_0005991. For cell culture, CAL27 cells were seeded in 6-well plates. Then, using Liposome 2000 (Invitrogen, United States) by the manufacturer’s instructions, siRNAs or controls (General, Anhui, China) were added to the cells at a final concentration of 50 nM.
10
Circulating ZNF609 Modulates Melanoma
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We evaluated the effect of circZNF609 on melanoma in vitro using SK-MEL-28 and A375 cells. The SK-MEL-28 and A375 cells were incubated at 37°C and 5% CO2 in FBS (10%, Hyclone, USA) and streptomycin (0.1 mg/mL, Hyclone, USA)/penicillin (100 units/mL, Hyclone, USA)-supplemented RPMI 1640 medium. The control shRNA, circZNF609 shRNA, pcDNA3.1-SIRT7, miR-138-5p mimic/inhibitor were synthesized (GenePharma, China). Liposome 2000 (Invitrogen, USA) was utilized for the transfection.
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