Small interfering rnas sirnas

Manufactured by Santa Cruz Biotechnology

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Small interfering RNAs (siRNAs) are a type of double-stranded RNA molecules that can trigger the degradation of specific target mRNA, thereby reducing the expression of the corresponding gene. siRNAs function by binding to and activating the RNA-induced silencing complex (RISC), which then cleaves the target mRNA.

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9 protocols using small interfering rnas sirnas

Cytotoxicity and Apoptosis Assays

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A lactate dehydrogenase (LDH) assay kit for cytotoxicity detection was purchased from Roche Applied Science (Risch-Rotkreuz, Switzerland). The APO-BrdU™ TUNEL assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Nuclear and Cytoplasmic Extraction Reagents (NE-PER) nuclear and cytoplasmic extraction reagents, a chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), and the MitoProbe™ JC-1 assay kit for flow cytometry were purchased from Thermo Fisher Scientific. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific. Gold chloride tri-hydrate, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and other reagents were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Small interfering RNAs (siRNAs) against NRF2 and NAD(P)H quinone dehydrogenase 1 (NQO1) and antibodies against α-tubulin, TATA-binding protein (TBP), NRF2, heme oxygenase-1 (HO-1), and NQO1 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Cleaved caspase-3, a cleaved caspase-3 (Asp175)–Alexa Fluor^® 488 antibody conjugate, p53, and p21 were purchased from Cell Signaling Technology (Beverly, MA, USA). The FuGENE HD transfection reagent and X-tremeGENE siRNA transfection reagent were obtained from Hoffmann-La Roche Ltd. (Basel, Switzerland).

Park S.Y., Chae S.Y., Park J.O., Lee K.J, & Park G. (2017). Kalopanacis Cortex extract-capped gold nanoparticles activate NRF2 signaling and ameliorate damage in human neuronal SH-SY5Y cells exposed to oxygen–glucose deprivation and reoxygenation. International Journal of Nanomedicine, 12, 4563-4578.

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EGFR Knockdown in HaCaT Cells

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Small-interfering RNAs (siRNAs) against human EGFR and a non-specific siRNA control were purchased from Santa Cruz Biotechnology. HaCaT cells were cultured to 50-60% confluence, and then transfected with the siRNA duplex using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s protocol. Non-specific siRNA was also transfected as a control.

Yoo Y.H., Kim Y.R., Kim M.S., Lee K.J., Park K.H, & Hahn J.H. (2014). YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR. BMB Reports, 47(10), 581-586.

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Isolation and Culture of Rat Renal Epithelial Cells

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Isolation and culture of rat primary tubular epithelial cells were performed as described previously [23 (link)]. Young and aging renal tubular epithelial cells were respectively isolated from young and old rat kidneys and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. After three passages, the cells were collected for experimentation. Small interfering RNAs (siRNAs) targeting Sod1, which is highly expressed in tubular epithelial cells, and a RNAi negative control duplex were purchased from Santa Cruz Biotechnology. The RNAi oligonucleotide and RNAi negative control duplex were transfected into cells as instructed by the manufacturer.

Li D., Zhao D., Zhang W., Ma Q., Liu D., Huang Q., Zheng Y., Bai X., Sun X, & Chen X. (2018). Identification of proteins potentially associated with renal aging in rats. Aging (Albany NY), 10(6), 1192-1205.

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Intracellular Signaling Pathway Analysis

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Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, 2 mM l-glutamine, lipofectamine 2000, and TRIzol were purchased from Invitrogen (Carlsbad, CA, USA). Cell culture dishes, 6-well, and 12-well plates were purchased from Greiner Bio-One (Frickenhausen, Germany). Polyvinyldifluoride (PVDF) membrane and an Immobilon Western Chemiluminescent HRP Substrate detection system were purchased from Millipore (Billerica, MA, USA). All of the primary antibodies specific for IL-6 (sc-28343), CXCR2 (sc-32780), c-Raf (sc-7267), MEK (sc-6250), ERK (sc-514302), JNK (sc-7345), p38 (sc-81621), c-Jun (sc-166540), and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal rabbit antibodies specific for phosphorylated forms of c-Raf (#9427), MEK (#3958), ERK (#4376), JNK (#9255), p38 (#9216), and c-Jun (#2361) were purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). All inhibitors against signal pathway components were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human CXCL1 was obtained from PeproTech (Rocky Hill, NJ, USA). All small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Hou S.M., Chen P.C., Lin C.M., Fang M.L., Chi M.C, & Liu J.F. (2020). CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK, and AP-1 pathway. Arthritis Research & Therapy, 22, 251.

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Transfecting Src, HIF-1α, and Ubiquitin

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HA-HIF-1α, HA-Ub, and pcDNA3-Src plasmids were purchased from Addgene (Watertown, MA, USA). Small interfering RNAs (siRNAs) targeting Src were purchased from Santa Cruz Biotechnology. Cells were transiently transfected with plasmids or siRNAs using Lipofectamine 2000 or RNA iMAX reagents (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Jo M.J., Kim B.G., Kim W.Y., Lee D.H., Yun H.K., Jeong S., Park S.H., Kim B.R., Kim J.L., Kim D.Y., Lee S.I, & Oh S.C. (2021). Cannabidiol Suppresses Angiogenesis and Stemness of Breast Cancer Cells by Downregulation of Hypoxia-Inducible Factors-1α. Cancers, 13(22), 5667.

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HSP Signaling Pathway Modulation

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Protein A/G PLUS-Agarose, anti-rabbit IgG-HRP secondary antibody, rabbit polyclonal antibodies against HSP90, HSP70, HSP27, p38 MAPK, JNK, ERK, Akt, HSF1 and β-actin, small interfering RNAs (siRNAs) against HSP90, HSP70 and HSP27 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The following rabbit polyclonal antibodies, including phospho-p38 MAPK (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and phospho-Akt (Ser473), were obtained from Cell Signaling and Neuroscience (Danvers, MA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Liu J.F., Chen P.C., Ling T.Y, & Hou C.H. (2022). Hyperthermia increases HSP production in human PDMCs by stimulating ROS formation, p38 MAPK and Akt signaling, and increasing HSF1 activity. Stem Cell Research & Therapy, 13, 236.

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Investigating ANGPTL2 and VEGF-A Signaling

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ANGPTL2 (ab199133) antibody was purchased from Abcam (Cambridge, MA, USA), VEGF-A (A17877) was purchased from Abclonal (Cambridge, MA, USA), p-p65 (3033) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA), β-actin (GT5512) and p65 (GTX102090) antibodies were purchased from GeneTex (Hsinchu, Taiwan). Small interfering RNAs (siRNAs) against integrin α5 (sc-29372) and β1(sc-35674), and immunoglobulin (Ig)-like transcript 4 (ILT4) (sc-45200) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). VEGF-A recombinant protein was acquired from PeproTech (Rocky Hill, NJ, USA). The p38 MAPK inhibitor SB203580 (HY-10256) and NF-κB inhibitor PDTC (P8765-1G) was acquired from Sigma-Aldrich (St. Louis, MO, USA). The ANGPTL2 overexpression plasmid (ANGPTL2 cDNA) was commercially synthesized by NCFB (Academia Sinica, Taiwan). ANGPTL2 (clone ID: TRCN0000158500) knockdown (sh-ANGPTL2) was purchased from the National RNAi Core Facility (RNAi Core, Academia Sinica, Taiwan).

Liu P.I., Jiang Y.J., Chang A.C., Huang C.L., Fong Y.C., Guo J.H., Liu C.L., Wang S.W., Liu J.F., Chang S.L, & Tang C.H. (2023). ANGPTL2 promotes VEGF-A synthesis in human lung cancer and facilitates lymphangiogenesis. Aging (Albany NY), 15(5), 1652-1667.

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Evaluating Gαs-PKA Signaling Pathways

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A long-form of stimulatory GTP-binding protein α subunit (Gαs) that contains constitutively active mutation and a Glu-Glu tag in the pcDNA3.1 + vector (GαsQL) was obtained from Missouri S&T cDNA Resource Center (Rolla, MO, USA). A dominant negative mutant PKA catalytic subunit (dnPKA) was provided by Dr. G. Stanley McKnight (University of Washington, WA, USA). The PKA catalytic subunit (GPKA) with a green fluorescent protein (GFP)-tag was obtained from Dr. Steven H Green (University of Iowa, IA, USA). Fluorescent reporter plasmids for NHEJ were the gift from Dr. Vera Gorbunova (University of Rochester, USA), and the pDsRed-N1 vector was obtained from Dr. MiOk Lee (Seoul National University, Seoul, Korea). Small interfering RNAs (siRNAs) against ataxia telangiectasia mutated (ATM, siATM) and protein phosphatase A (PP2A) B56δ (siB56δ) and control siRNAs were obtained from Santa Cruz Biotechnology. A short hairpin RNA (shRNA) that targets PKA and the control shRNA were bought from Sigma-Aldrich. The expression plasmids and RNAs were transiently transfected using Lipofectamine 3000 (Invitrogen, CA, USA).

Noh S.E, & Juhnn Y.S. (2020). Inhibition of non-homologous end joining of gamma ray-induced DNA double-strand breaks by cAMP signaling in lung cancer cells. Scientific Reports, 10, 14455.

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Transfection of miR-375 and siRNA in GC cells

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TINCR sequences were cloned into pcDNA.3.1 vector. We conducted the transfections by using a Lipofectamine 2000 kit (Thermo Fisher Scientific). Human GC HGC-27 and SNU-1 cell lines were transfected with miR-375 mimic, inhibitor, and negative control, which were purchased from GenePharma (Shanghai, China). miR-375 mimic, inhibitor, and negative control at a final concentration 50 nM were transfected using Lipofectamine^® 2000 into cells seeded onto six-well plates. Twenty-four hours after transfection, cells were analysed as required. Small interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Transfection of siRNA duplexes into GC cells was carried out using Lipofectamine 2000 according to the manufacturer’s instructions. At 24 h posttransfection, cells were harvested using real-time PCR (polymerase chain reaction) or Western blot analysis.

Chen Z., Liu H., Yang H., Gao Y., Zhang G, & Hu J. (2017). The long noncoding RNA, TINCR, functions as a competing endogenous RNA to regulate PDK1 expression by sponging miR-375 in gastric cancer. OncoTargets and therapy, 10, 3353-3362.

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