Scrambled and pcDNA-3.1-14-3-3η-Flag were synthesized by Generay Biotech (Shanghai, China); while 14–3-3η- and NF-κB/p65 siRNA were purchased from Santa Cruz Biotechnology (http://datasheets.scbt.com/sc-43581.pdf , and https://datasheets.scbt.com/sc-29410.pdf ) [12 (link), 16 (link)]. Cells were transiently transfected via lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA), according to the manufacturer’s protocol (Note: plasmids used: 5 ng/ml. siRNAs used: 20 nM). After transfection, the cells were cultured in fresh medium supplemented with 10% FBS for another 24 h before being used for other experiments.
Nf κb p65 sirna
Manufactured by Santa Cruz Biotechnology
Sourced in China, United States
NF-κB p65 siRNA is a small interfering RNA (siRNA) targeting the p65 subunit of the NF-κB transcription factor. It is designed to knockdown the expression of NF-κB p65 in cells.
Automatically generated - may contain errors
Lab products found in correlation
Control vector, by GenePharma (2 mentions)
Puromycin, by Santa Cruz Biotechnology (2 mentions)
Pinx1 sirna, by GenePharma (2 mentions)
Pinx1 shrna expression vector, by GenePharma (2 mentions)
Silentfect lipid reagent, by Bio-Rad (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Non specific control sirna, by GenePharma (1 mentions)
Scrambled sirna, by GenePharma (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Stat3 sirna, by Santa Cruz Biotechnology (1 mentions)
Hdac1 sirna, by Santa Cruz Biotechnology (1 mentions)
Interferin, by Polyplus Transfection (1 mentions)
Human tnf α, by R&D Systems (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Mirna inhibitor, by Qiagen (1 mentions)
Anti human tnf α antibody, by R&D Systems (1 mentions)
Mirna mimic, by Qiagen (1 mentions)
Egm 2 singlequot, by Lonza (1 mentions)
10 protocols using nf κb p65 sirna
1
Overexpression and Silencing of Key Proteins
2
Modulating NF-κB p65 in MHCC-97H Cells
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NF-κB p65 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China). Human MHCC-97H cells were transiently transfected with p65 siRNA or with a p65 cDNA plasmid (kindly gifted by Dr. Chen), using Lipofectamine 2000 and following the manufacture’s instruction. After the MHCC-97H cells were transfected with p65 siRNA or p65 cDNA for 6 hours, the cells were exposed to TPL (25 nM) for 48 hours.
3
PinX1 Overexpression and Knockdown Cell Lines
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Cells were grown to 50% confluence before small interfering RNA (siRNA) transfection. PinX1 siRNA (GenePharma, Shanghai, China), NF-κB-p65 siRNA (Santa Cruz, CA, USA) or Nonspecific control siRNA (GenePharma, Shanghai, China) was transfected by siLentFect Lipid Reagent (Bio-Rad, CA, USA) according to the manufacturer’s protocol. The pEGFP-C3 and pEGFP-C3-PinX1 expression plasmids were obtained from Dr Xiao-Fen Lai (Southern Medical University, Guangzhou, China). Transfection of the pEGFP-C3-PinX1 plasmid and the pEGFP-C3 vector into the MDA-MB-231 and BT-549 cells were carried out using Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s instructions.
PinX1 overexpression MDA-MB-231 cell lines (PinX1OE-MDA-MB-231), PinX1 knockdown MDA-MB-231 cell lines (PinX1KD-MDA-MB-231) and control MDA-MB-231 cell lines (Ctrl-MDA-MB-231) were established by infecting with lentivirus packing PinX1 expression vector, PinX1 shRNA expression vector and control vector respectively (GenePharma, Shanghai, China). It is the same as the construction of PinX1 overexpression BT-549 cell lines (PinX1OE-BT-549), PinX1 knockdown BT-549 cell lines (PinX1KD-BT-549) and control BT-549 cell lines (Ctrl-BT-549). Target cells were infected with lentivirus for 48 hours then selected with puromycin (Santa Cruz) for three weeks.
PinX1 overexpression MDA-MB-231 cell lines (PinX1OE-MDA-MB-231), PinX1 knockdown MDA-MB-231 cell lines (PinX1KD-MDA-MB-231) and control MDA-MB-231 cell lines (Ctrl-MDA-MB-231) were established by infecting with lentivirus packing PinX1 expression vector, PinX1 shRNA expression vector and control vector respectively (GenePharma, Shanghai, China). It is the same as the construction of PinX1 overexpression BT-549 cell lines (PinX1OE-BT-549), PinX1 knockdown BT-549 cell lines (PinX1KD-BT-549) and control BT-549 cell lines (Ctrl-BT-549). Target cells were infected with lentivirus for 48 hours then selected with puromycin (Santa Cruz) for three weeks.
4
Knockdown of NF-κB p65 in MDA-MB-231 Cells
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NF-κB p65 siRNA was purchased from Santa Cruz Biotechnology. Briefly, 2 × 10 5 MDA-MB-231 cells were plated in 6-well plates and transfected with 1 μg of p65 siRNA or control siRNA oligos using lipofectamine 2000 as per the manufacturer’s instructions. Cells were incubated for 24 hours after which cells were harvested and analyzed for p65 and FABP5 expression using Western blot analysis.
5
Establishment and Characterization of PinX1 Cell Lines
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The pEGFP-C3 and pEGFP-C3-PinX1 expression plasmids were obtained from Dr Xiao-Fen Lai (Southern Medical University, Guangzhou, China). The PinX1 siRNA and scrambled siRNA were purchased from GenePharma (Shanghai, China). NF-κB-p65 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Transfection of the pEGFP-C3-PinX1 plasmid and the pEGFP-C3 vector into the 786-O and ACHN cells were carried out using Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer's protocol. PinX1 siRNA, NF-κB-p65 siRNA or scrambled siRNA was transfected into the 786-O and ACHN cells by siLentFect Lipid Reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. The PinX1 overexpression 786-O cell lines (PinX1OE-786-O), PinX1 knockdown 786-O cell lines (PinX1KD-786-O) and control 786-O cell lines (Ctrl-786-O) were established by infecting with lentivirus packing PinX1 expression vector (EGFP is not fused with PinX1 in this vector), PinX1 shRNA expression vector and control vector respectively (GenePharma, Shanghai, China). Target cells were infected with lentivirus for 48 hours then selected with puromycin (Santa Cruz) for 3 weeks.
6
NF-κB p65 Silencing in BMDMs
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To silence NF-κB p65, BMDMs were transfected with control siRNA or NF-κB p65siRNA (100 nM; Santa Cruz Biotechnology Inc., Dallas, TX, USA; sc-37007 and sc-29411, respectively) using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
7
Knockdown of STAT3 and NF-κB in A-549 Cells
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For the study, VCAM-1 siRNA, STAT3 siRNA, NF-κB (p65) siRNA or STAT3 siRNA along with respective scramble control were procured from Santa Cruz biotech, USA. The transfecting the cells with siRNA, the A-549 cells were trypsinized in suspension and 3×105 cells were transferred in 6 well plates. The cells received transfection of STAT3 siRNA and NF-κB siRNA following the manufacturers protocol, the cells were collected after defined time intervals for further study. Western blot analysis was done for confirming the transfection.
8
Silencing NF-κB Pathway in Breast Cancer Cells
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NF-κB p65 was silenced in BT474 and SKBR3 cells using NF-κB p65 siRNA (Santa Cruz, Heidelberg,Germany). IKKα or IKKβ was silenced in BT474 and SKBR3 cells using IKK siRNA (Santa Cruz, Heidelberg, Germany). Non-targeted siRNA (Santa Cruz, Heidelberg, Germany) was used a control. BT474 and SKBR3 cells were transiently transfected with NF-κB p65 siRNA or scrambled siRNA according to the manufacturer’s instructions and 24 h post-transfection, cells were either collected for Western blot analysis or treated with plumbagin.
9
Regulation of miR-146a expression
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The pGL3-miR-146a promoter reporter plasmid (miR-146a promoter-luc) was a pGL-basic vector containing a 560–base pair upstream sequence of the predicted transcriptional start site of the pri-miR-146a (primary transcript of miR-146a; Taganov et al., 2006 (link)). The NF-κB p65 submit overexpression vector was a kind gift from Jae Won Rhee (Rhee et al., 2007 (link)). The kappa B (IκB) dominant-negative (IκB DN, IκB (S32A⁄S36A)) plasmid was used to inhibit NF-κB activity (Tang et al., 2009b (link)). MiR-146a mimics (double-stranded RNA oligonucleotides) and inhibitors (single-stranded chemically modified oligonucleotides) (GenePharma, Shanghai, China) were used to overexpress and inhibit miR-146a expression in cells. Negative control mimics or inhibitors (GenePharma, Shanghai, China) were transfected to act as matched controls. HDAC1 siRNA (sc-35422, Santa Cruz Biotechnology, Santa Cruz, CA) and NF-κB p65 siRNA (sc-37007, Santa Cruz Biotechnology) were used to inhibit the activity of HDAC1 and NF-κB p65 submit, respectively. Macrophages described in the Cell culture and treatment section were transfected with RNAs at a final concentration of 10 nM using INTERFERin (Polyplus-Transfection SA, Illkirch, France) according to the manufacturer’s instructions.
10
Isolation and Transfection of MNCs from Blood
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MNCs were isolated from human whole venous blood and cord blood by conventional Ficoll-Hypaque density gradient centrifugation and resuspended in EGM-2 SingleQuots (#CC-4176, Lonza, Basel, Switzerland). Peripheral blood or cord blood MNCs (PBMNCs or CBMNCs, respectively, 4 × 106 cells/mL) were seeded on a fibronectin (50 μg/mL; Sigma-Aldrich)-coated plate and incubated in a 5% CO2 incubator at 37 °C for 4 days. After being carefully washed, adherent MNCs were transfected with or without AllStars Negative Control for siRNA and miRNA mimic experiments (#1027280, Qiagen), miScript Inhibitor Negative Control for miRNA inhibitor experiments (#1027272, Qiagen), miRNA inhibitors (80 nM, Qiagen), miRNA mimics (80 nM, Qiagen), and NF-κB p65 siRNA (80 nM, Santa Cruz) as described previously12 (link)–15 , followed by treatment with or without human TNF-α (10 ng/mL, R&D Systems) for another 4 days. In addition, some adherent MNCs were cultured in medium 199 supplemented with 30% human autologous serum in the presence or absence of an anti-human TNF-α antibody (5 μg/mL; #MAB210, R&D Systems) for another 4 days.
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