Sinrf2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

SiNrf2 is a small interfering RNA (siRNA) product designed to target the Nrf2 (nuclear factor erythroid 2-related factor 2) gene. Nrf2 is a transcription factor that regulates the expression of genes involved in the cellular response to oxidative stress. The SiNrf2 product is intended to modulate the activity of the Nrf2 pathway, but its specific applications and intended uses are not within the scope of this factual description.

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12 protocols using sinrf2

Molecular Mechanisms of Nrf2 and ERK Regulation

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Gln, H₂O₂, LPS, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies against iNOS, Nrf2, HO-1, ERK, phospho-ERK (p-ERK), and β-actin, and silencing RNA (siRNA) for Nrf2 (siNrf2), ERK (siERK), and control siRNA (siCON) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-labeled goat anti-rabbit immunoglobulin was purchased from KOMA Biotechnology (Seoul, Korea). Cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP) were obtained from Tocris Bioscience (Bristol, UK) and PD98059 was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and antibiotics were obtained from WelGENE Inc. (Daegu, Korea). Other chemicals were purchased from Sigma-Aldrich Chemical Co.

Jayasooriya R.G., Molagoda I.M., Dilshara M.G., Choi Y.H, & Kim G.Y. (2020). Glutamine Cooperatively Upregulates Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglial Cells through the ERK and Nrf-2/HO-1 Signaling Pathway. Antioxidants, 9(6), 536.

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Molecular Mechanism of Nrf2 Regulation

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Rhodamine 123, doxorubicin, verapamil, and tert-butylhydroquinone (tBHQ) were purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies against human P-gp, Keap1 and Nrf2 were purchased from Abcam, Inc. (Cambridge, MA). Primary antibodies against human β-actin and histone H3, siNrf2, and siControl were obtained from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Lipofectamine 2000 and TRIZOL were obtained from Invitrogen (Carlsbad, CA). All other chemicals and solvents used were of analytical reagent grade or better.

Yang X., Hu G., Lv L., Liu T., Qi L., Huang G., You D, & Zhao J. (2019). Regulation of P-glycoprotein by Bajijiasu in vitro and in vivo by activating the Nrf2-mediated signalling pathway. Pharmaceutical Biology, 57(1), 184-192.

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Nrf2 Silencing in PC12 Cells

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Control siRNA (siCtr) and siRNA targeting Nrf2 (siNrf2, cat# sc-156128) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PC12 cells were transfected with siCtr or siNrf2 using the Lipofectamine 2000 transfection reagent and Opti-MEM I reduced serum medium (Thermo Scientific), as reported previously [42 (link),43 (link)]. Briefly, cells were incubated in media without antibiotic–antimycotic until reaching 70–90% confluency. Opti-MEM I and siRNA were mixed to make solution A, and Opti-MEM I and Lipofectamine 2000 were mixed to make solution B. The two solutions were then left in situ for 5 min and gently mixed to yield solution C. After 20 min, the culture media were replaced with fresh media without antibiotics containing solution C at a final concentration of 100 nM siRNA. After 6 h of transfection, the media containing siRNA were exchanged with fresh media containing antibiotic–antimycotic. After 36–48 h with daily media exchanges, the cells were used for further experiments.

Gouda N.A, & Cho J. (2022). Omarigliptin Mitigates 6-Hydroxydopamine- or Rotenone-Induced Oxidative Toxicity in PC12 Cells by Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Actions. Antioxidants, 11(10), 1940.

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Cellular Transfection for Hypoxia-Reoxygenation Study

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L02 cells were transfected with si-FGFR3 (Santa Cruz, sc-29314) or si-NRF2 (Santa Cruz, sc-37030) or siRNA scrambled (negative) control (si-scramble; Santa Cruz, sc-37007) by using Lipofectamine 2000 for 6 h in Opti-MEM. HepG2 cells were transfected with si-USP16 (Limibio Co., Ltd) by using Lipofectamine 2000 for 6 h in Opti-MEM. LX-2 cells were transfected with si-FGF18 (Santa Cruz, sc-39478) by using Lipofectamine 2000 for 6 h in Opti-MEM. After transfection, L02 cells, HepG2 cells and LX-2 cells were subjected to H/R challenge. All the primers for si-RNA sequence used in this study are shown in the Supplementary Data 5.

Tong G., Chen Y., Chen X., Fan J., Zhu K., Hu Z., Li S., Zhu J., Feng J., Wu Z., Hu Z., Zhou B., Jin L., Chen H., Shen J., Cong W, & Li X. (2023). FGF18 alleviates hepatic ischemia-reperfusion injury via the USP16-mediated KEAP1/Nrf2 signaling pathway in male mice. Nature Communications, 14, 6107.

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siRNA Transfection in MRC5 Cells

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Predesigned siRNAs (100 pmol) were transfected into MRC5 cells with Lipofectamine 3000 reagent (Cat#L3000015, Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. We used the following siRNAs: siRNA negative control (Cat#sc-37007, Santa Cruz), sip62 (Cat#1144479, Bioneer, Daejeon, Korea), siNrf2 (Cat#sc-37030, Santa Cruz), siATF6 (Cat#sc-37699, Santa Cruz), siHSF1 (Cat#sc-35611, Santa Cruz), siHIF1 (Cat#sc-35561, Santa Cruz), and siRTN4 (Cat#sc-43974, Santa Cruz).

Kang J.I., Kim D.H., Sung K.W., Shim S.M., Cha-Molstad H., Soung N.K., Lee K.H., Hwang J., Lee H.G., Kwon Y.T, & Kim B.Y. (2021). p62-Induced Cancer-Associated Fibroblast Activation via the Nrf2-ATF6 Pathway Promotes Lung Tumorigenesis. Cancers, 13(4), 864.

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SH-SY5Y Cell-based Differentiation and Neuroprotection

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SH-SY5Y cells were purchased from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's modified Eagle medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated horse serum (Gibco), 5% heat-inactivated fetal calf serum (Gibco), 100 IU/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator containing 5% CO₂ at 37°C. SH-SY5Y cells were treated with 50 ng/ml 2.5S nerve growth factor (Promega, Madison, WI, USA) for 9 days to induce differentiation. Cells were plated in 6-well plates and passaged at 60–70% confluence. In the experiments, cells were pretreated with 25–100 μM Sal for 24 h and then exposed to 500 μM MPP+ for an additional 24 h. In some experiments, cells were transfected with siNrf2 or siDJ-1 (Santa Cruz Biotechnology) using lipofectamine 2000 transfection reagent 2–4 days before the treatment of Sal and MPP+.

Wu L., Xu H., Cao L., Li T., Li R., Feng Y., Chen J, & Ma J. (2017). Salidroside Protects against MPP+-Induced Neuronal Injury through DJ-1-Nrf2 Antioxidant Pathway. Evidence-based Complementary and Alternative Medicine : eCAM, 2017, 5398542.

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Nrf2 Modulation in Oxalate-Induced MDCK Cells

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The small interfering RNA (siRNA) duplexes against Nrf2 (si-Nrf2) (Santa Cruz Biotechnology) and control siRNA (si-Control) (Santa Cruz Biotechnology) were transfected to MDCK cells. Briefly, the cells seeded in 6-well plate were transfected with 80 pmol si-Nrf2 or si-Control mixed with siRNA transfection reagent in the transfection medium (Santa Cruz Biotechnology) according the manufacturer’s instructions. The transfected cells were then incubated in a humidified incubator with 5% CO₂ at 37 °C for 6 h. After 24-h post-transfection, the cells were subjected to oxalate and EGCG treatments using the same protocols as for the non-transfected cells as detailed above. Expression levels of Nrf2, ZO-1 and vimentin were examined by immunofluorescence staining, whereas catalase level was evaluated by Western blotting as aforementioned.

Kanlaya R., Khamchun S., Kapincharanon C, & Thongboonkerd V. (2016). Protective effect of epigallocatechin-3-gallate (EGCG) via Nrf2 pathway against oxalate-induced epithelial mesenchymal transition (EMT) of renal tubular cells. Scientific Reports, 6, 30233.

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Nrf2 Silencing and BDB Treatment

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The siRNA constructs used were a mismatched siRNA control (siControl, Santa Cruz Biotechnology) and a siRNA against Nrf2 (siNrf2, Santa Cruz Biotechnology). The cells were transfected with 10–50 nM siRNA using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction. At 24 h after transfection, the cells were treated with 10 μM of BDB for 24 h and subjected to a western blot analysis.

Kim K.C., Hyun Y.J., Madduma Hewage S.R., Piao M.J., Kang K.A., Kang H.K., Koh Y.S., Ahn M.J, & Hyun J.W. (2017). 3-Bromo-4,5-dihydroxybenzaldehyde Enhances the Level of Reduced Glutathione via the Nrf2-Mediated Pathway in Human Keratinocytes. Marine Drugs, 15(9), 291.

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Knockdown of PGC-1α and Nrf2 in H9c2 Cells

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The small interfering RNA (siRNA) duplexes corresponding to rat PGC-1α (siPGC-1α), Nrf2 (siNrf2) and the negative control siRNA (siNC) were purchased from Santa Cruz Biotechnology, Inc. siRNAs (100 nM) were transfected into H9c2 cells using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. In brief, H9c2 cells (5×10⁵) were plated in a 6-well plate and cultured for 24 h. When the cells reached 80% confluence, Lipofectamine 2000 was added to the medium without serum and incubated for 5 min at room temperature. The diluted siRNA was gently mixed with the medium containing Lipofectamine 2000 and incubated for 20 min at room temperature prior to adding the mixture to each well containing cells and medium. The transfected cells were cultured at 37°C in a CO₂ incubator for 24 h prior to subsequent experiments. Untransfected cells were used as a blank control, and cells transfected with siNC were used as a negative control.

Zhi W., Li K., Wang H., Lei M, & Guo Y. (2020). Melatonin elicits protective effects on OGD/R-insulted H9c2 cells by activating PGC-1α/Nrf2 signaling. International Journal of Molecular Medicine, 45(5), 1294-1304.

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Chondrocyte Nrf2 Silencing Protocol

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The si-Nrf2 and si-NC (negative control) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The chondrocytes were seeded in a six-well plate and cultured for 24 h until they were 50–70% confluent. Then, the cells were transfected with 50 nM si-NC or si-Nrf2 using Lipofectamine 2000 siRNA transfection reagent (Thermo Fisher) for 48 h.

Shao Z., Pan Z., Lin J., Zhao Q., Wang Y., Ni L., Feng S., Tian N., Wu Y., Sun L., Gao W., Zhou Y., Zhang X, & Wang X. (2020). S-allyl cysteine reduces osteoarthritis pathology in the tert-butyl hydroperoxide-treated chondrocytes and the destabilization of the medial meniscus model mice via the Nrf2 signaling pathway. Aging (Albany NY), 12(19), 19254-19272.

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