Pcdna3.1 v5 his topo ta expression kit

Primacy NPCs were isolated from human intervertebral disc (IDD patients and controls), mouse intervertebral disc (miR-150-5p KO and C57BL/6 wild-type mice). NPCs were preserved as a monolayer in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FCS), 100 IU/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% carbon dioxide at 37 °C. All the experiments involved the use of human NPCs at a confluence rate of 85%. As previously described [10 (link)], the cells were inoculated into 96-well plates for three-dimensional culture of the NPCs. The NPCs were then cultured at 37 °C and 5% carbon dioxide in 200 µl of NPC growth medium. After 21 days of culture, the cells were collected for further evaluation. Using a Silencer® siRNA labeling kit or miRNA negative control (mirVana miRNA mimics/inhibitor negative control #1) (Life Technologies), Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA), and Cy3-labeled or unlabeled miR-150-5p (a mirVana miRNA mimic or inhibitor) were transfected into human NPCs, SW1353 cells, or C28/I2 cells at 50 nM. The FBXW11 expression plasmid (pcDNA 3.1/V5-His TOPO TA Expression Kit) (Invitrogen) was subsequently obtained.

Full-length E6 of HPV16 was amplified by PCR from CasKi cells containing the viral genome of HPV16. The resulting PCR products were purified with a GENECLEAN III kit (MP Biomedicals, Vista, CA). Purified fragments were cloned into a eukaryotic expression vector, pcDNA3.1/V5-His TOPO TA expression kit (Invitrogen, Carlsbad, CA), and the resultant recombinants were transfected into HPV non-infected lung cancer cell lines. On the day prior to transfection, the cells were seeded at 1 × 10⁵ cells per well. After incubation overnight, the cells (30%–50% confluent) were washed twice with phenol red-free DMEM or RPMI 1640 medium without FBS. After incubation with 1 mL of phenol red-free DMEM or RPMI 1640 medium with 10% FBS for 3 h, calcium chloride-Hepes-buffer saline and recombinant DNA solution were added dropwise to the medium in each well of the plate. The cells were then returned to the incubator. After 4 h, the medium was aspirated and the cells were shocked with glycerol solution for 30 sec and then washed twice with PBS. Stable transfectants were selected by culturing transfected cells in the medium containing the antibiotic G418.

The murine SphK1 gene was amplified using TaKaRa Ex Taq Hot Start Version (TaKaRa, Kusatsu, Japan). The SphK1-expressing plasmid was then constructed using the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen, Carlsbad, CA, USA). sCA-encapsulated plasmids were prepared as described previously [27 (link),47 (link)]. Thus, 4 μL of 1 M CaCl₂ was incubated at 37 °C for 30 min with 2 μg of plasmid DNA in 1 mL of an inorganic solution (NaHCO₃, 44 mM; NaH₂PO₄, 0.9 mM; CaCl₂, 1.8 mM; pH 7.5) and then centrifuged at 12,000 rpm for 3 min. After the pellet was dissolved with DMEM, the solution was sonicated in a water bath for 10 min, thus generating sCA-encapsulated plasmids. Cells cultured in 6-well plates for 24 h were incubated with the plasmid-sCA-DMEM solution for 6 h. The medium was then replaced with DMEM containing 10% FBS. After another 48 h, the cells were collected for total RNA isolation. The plasmid-sCA ointment was generated by dissolving the sCA pellet with 50 µL of PBS, mixing it with 100 µg of plasmid DNA, and then mixing the solution into 200 µL of Aquaphor^®. The four wounds of two mice were each treated once with 250 µL of ointment.

The cultured primary human NP cells were transfected with mimics or inhibitor labeled or unlabeled with Cy3 using the Silencer® siRNA Labeling Kit (#AM1636) or miRNA negative control (mirVanaTM miRNA mimics/inhibitor Negative Control #1, catalog number: 4464061/4464079) (Life Technologies) at 50 nM using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen). To suppress SIRT1 expression, cells were transfected with either SIRT1 siRNA or control scrambled siRNA (Thermo Scientific Dharmacon®) using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The SIRT1 expression plasmid (pcDNA™3.1/V5-His TOPO™ TA Expression Kit) was obtained (Invitrogen™). At 48 h after transfection, the cellular lysates were collected to analyze the expression of genes of interest.

Androgen-insensitive LNCaP cells and CHO cells were cultured in RPMI 1640 and DMEM/F-12 medium, respectively. These culture media were also supplemented with 10% v/v fetal bovine serum (FBS), 2 mM L-glutamine, 0.2% w/v sodium bicarbonate, and 1% v/v penicillin-streptomycin. Cultures were maintained at 37°C and 5% CO₂ until cells reached 60–70% confluence. Apoptosis was induced by incubating cells with culture medium deprived of FBS for different periods of time. Ionomycin was prepared as 5 mM stock solutions in DMSO. Ionomycin at a final concentration of 10 µM was applied to CHO and LNCaP cells cultures and incubated for different periods of time. Both cell lines were transfected with pcDNA3.1 ARP2 V5-His (Transfection efficiencies: TE/LNCaP 32%; TE/CHO 46%) and pcDNA3.1/V5-His-TOPO lacZ plasmids (TE/LNCaP 43%; TE/CHO 54%) to induce transient expression of ARP2-V5 and β galactosidase-V5, respectively. For normalization of cell viability assays, both cell lines were also transfected with plasmid pcDNA3.1 V5-His (without arp2 cDNA) (TE/LNCaP 68%; TE/CHO 80%). CHO cells were transfected with pEGFP-N1 (TE 82%) and pcDNA3.1 ARP2-eGFP V5-His plasmids (TE 50%) to obtain eGFP and ARP2-eGFP expression, respectively. Transfections were performed using Lipofectamine 2000 as described in the pcDNA3.1/V5-His TOPO TA Expression Kit insert from Invitrogen (Carlsbad, CA, USA).

The VSV-G expression vector pMDG, gag-pol expression vector pCMVD8.91, and pLKO.1 encoding the AhR small hairpin RNA (shRNA) (TRCN0000245286; RNAi core, Taipei, Taiwan) with the target sequence 5′-CCGGCCCACAACAATATAATGTC TTCTCGAGAAGACATTATATTGTTGTGGGTTTTT-3′, were co-transfected into 293 T cells by calcium phosphate precipitation. All plasmids were obtained from the National RNAi core facility (Academic Sinica, Taiwan). The virus-containing supernatants were collected after 24–48 h of transfection and were filtered through a 0.45-μm syringe filter. The viral supernatants were added to the A549 culture in the presence of 10 μg/mL polybrene (hexadimethrine bromide; 107689; Sigma, St. Louis, MO, USA). After 72 h of infection, lentivirus-infected A549 cells were used subsequent studies. Overexpression of human AhR was achieved by transfection with pcDNA3.1-AhR, which contained the full-length AhR gene (GenBank Accession number 196, NCBI) and was constructed using the pcDNA™3.1/V5-His TOPO^® TA expression kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. Transient transfection of cells was performed using Turbofect™ (Thermo Fisher) according to the manufacturer’s instructions to obtain AhR-overexpressing cells.

Total RNA was collected from the peripheral blood leucocytes of the patient by standard Trizol method. The full-length H229Q VDR cDNA was obtained by reverse transcription of total RNA using the M-MLV RT-PCR kit with primers 5'-CCGGCCGGACCAGAAGCCTT-3' and 5'-CTGCTGAGTAGCCGCCAGCC-3' followed by subcloning into the pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen). The wild-type (WT) VDR expression plasmid was generated by using the H229Q plasmid as a template with Quickchange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) and single primer (5'-CTCTCCATGCTGCCCCACCTGGCTGACCTGGTCAG-3') method[7 (link)]. All plasmids were completely sequenced for correctness.