Atg5 sirna

Manufactured by Santa Cruz Biotechnology

Sourced in United States

ATG5 siRNA is a short interfering RNA (siRNA) that targets the ATG5 gene, which is involved in the autophagy process. Autophagy is a cellular mechanism responsible for the degradation and recycling of damaged or unnecessary cellular components. The ATG5 siRNA can be used to study the role of autophagy in various cellular processes and disease models.

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10 protocols using atg5 sirna

siRNA Transfection for HT22 Cells

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For siRNA transfection, HT22 cells were plated in 60 mm dishes at 5 x
10⁵ cells/dish and 20 pmol ATG5 siRNA (#sc-41446 mouse; #sc41445-
human) or HIF1α siRNA (#sc-35562) from Santa Cruz Biotechnology or
control siRNA (#1027280) from Qiagen, were used along with RNAi max (Invitrogen)
according to the manufacturer’s instructions.

Soriano-Castell D., Currais A, & Maher P. (2021). Defining a Pharmacological Inhibitor Fingerprint for Oxytosis/Ferroptosis. Free radical biology & medicine, 171, 219-231.

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ATG5 siRNA Knockdown in HT22 Cells

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For siRNA transfections, HT22 cells were plated in 60 mm dishes at 5 × 10⁵ cells/dish. For transfection, 166 pmol ATG5 siRNA (#sc-41446) or control siRNA (#sc-37007), all from Santa Cruz Biotechnology (Santa Cruz, CA) was used along with RNAiMAX (Invitrogen) according to the manufacturer's instructions.

Dafre A.L., Schmitz A.E, & Maher P. (2019). Hyperosmotic Stress Initiates AMPK-Independent Autophagy and AMPK- and Autophagy-Independent Depletion of Thioredoxin 1 and Glyoxalase 2 in HT22 Nerve Cells. Oxidative Medicine and Cellular Longevity, 2019, 2715810.

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Targeted siRNA Delivery for Liver Protection

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ATG5 siRNA (Santa Cruz, CA, USA) was premixed with mannose-conjugated polymers (Polyplus transfection, USA) at a ratio specified by the manufacture and was administered by tail vein injection (siRNA 2 mg/kg) 4 h prior to the TAA administration as described previously (23 (link)).

Zhou S., Gu J., Liu R., Wei S., Wang Q., Shen H., Dai Y., Zhou H., Zhang F, & Lu L. (2018). Spermine Alleviates Acute Liver Injury by Inhibiting Liver-Resident Macrophage Pro-Inflammatory Response Through ATG5-Dependent Autophagy. Frontiers in Immunology, 9, 948.

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Knockdown of Innate Immune Sensors

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HL-60 and THP-1 cells were infected with MISSION lentivirus carrying pLKO.1 puro with shRNA constructs (STING-1: TRCN0000164628; STING2: TRCN0000135555; AIM2-1: TRCN0000107501; AIM2-2: TRCN0000107502; and cGAS: TRCN0000149984), which were purchased from Sigma-Aldrich. Each cell line infected with MISSION lentivirus carrying pLKO.1 puro and with a non-mammalian shRNA control construct was used as a shControl. Stably transfected cells were selected in culture medium supplemented with 1 μg/ml puromycin for more than 2 weeks. For the transient KD of ATG5 gene expression, HL-60 cells were transfected with ATG5 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) using GENOMONE-Si transfection reagent (Ishihara Sangyo, Osaka, Japan). As a siControl, cells were transfected with control siRNA-A (Santa Cruz Biotechnology). The KD efficiency of each gene was confirmed using quantitative real-time PCR (qRT-PCR) and western blotting analyses.

Baba T., Yoshida T., Tanabe Y., Nishimura T., Morishita S., Gotoh N., Hirao A., Hanayama R, & Mukaida N. (2021). Cytoplasmic DNA accumulation preferentially triggers cell death of myeloid leukemia cells by interacting with intracellular DNA sensing pathway. Cell Death & Disease, 12(4), 322.

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ATG5 Knockdown in HT22 Cells

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For siRNA transfections, HT22 cells were plated in 60 mm dishes at 5 × 10⁵ cells/dish and 166 pmol ATG5 siRNA (#sc-41446) or control siRNA (#sc-37007), all from Santa Cruz Biotechnology (Santa Cruz, CA), were used along with RNAiMAX (Invitrogen) according to the manufacturer’s instructions.

Dafre A.L., Schmitz A.E, & Maher P. (2017). Methylglyoxal-induced AMPK activation leads to autophagic degradation of thioredoxin 1 and glyoxalase 2 in HT22 nerve cells. Free radical biology & medicine, 108, 270-279.

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Cholesterol Efflux Regulation via AMPK-Dependent Autophagy

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RPMI 1640 medium FBS (C0252) was acquired from Beyotime Biotechnology (Shanghai, China). Phorbol‐12‐Myristate13‐acetate (PMA; P1585) and 3‐methyladenine (3MA, 5 mmol/L; M9281) were purchased from Sigma‐Aldrich. RACK1 siRNA (sc‐36354), ATG5 siRNA (sc‐41445), and primary antibodies such as RACK1 (sc‐17754), beclin‐1 (sc‐11427), LC3 (sc‐398822), ABCA1 (sc‐53482), GAPDH (sc‐47724), β‐actin (sc‐47778) and AMPK (sc‐17754) were acquired from Santa Cruz Biotechnology. Other primary antibodies like anti‐AMPK (2793) and anti–phospho‐AMPK Thr172 (2535) from acquired from Cell Signaling Technology, while p62 primary antibody was supplied by R&D Systems. Acetylated LDL (Ac‐LDL; L8940), FGF21 (ab63277) and secondary goat anti‐mouse IgG‐HRP (ab97023) and goat anti‐rabbit IgG (ab205718) antibodies came from Abcam, while pEGFP‐LC3 was supplied by Cell Biolabs.

Xiaolong L., Dongmin G., Liu M., Zuo W., Huijun H., Qiufen T., XueMei H., Wensheng L., Yuping P., Jun L, & Zhaolin Z. (2020). FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation. Journal of Cellular and Molecular Medicine, 24(9), 4992-5006.

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Beclin and Atg5 Silencing in LLC-PK1 Cells

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LLC-PK1 cells were seeded in a six-well plate with 5% FBS in M199 medium without antibiotics. Beclin siRNA (sc–29798) and Atg5siRNA (sc–41446) were obtained from Santa Cruz Biotechnology. Cells at 70% confluence were untreated (control) or transfected with the corresponding siRNA pools using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were treated as indicated and a portion of the samples were collected to analyze gene silencing by Western blot analysis.

Chandrika B.B., Yang C., Ou Y., Feng X., Muhoza D., Holmes A.F., Theus S., Deshmukh S., Haun R.S, & Kaushal G.P. (2015). Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury. PLoS ONE, 10(10), e0140025.

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Fibroblast and HeLa Cell Culture Protocols

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Wild-type mouse embryonic fibroblasts, TBK1^+/- mouse embryonic fibroblasts, TBK1^-/- mouse embryonic fibroblasts, and HeLa cervical cancer cells were maintained in DMEM growth medium consisting of DMEM supplemented with 10% fetal bovine serum (FBS; Life Technologies; 16010–159) and antibiotic/antimycotic (Life Technologies; 15240–062).
Bafilomycin A1 (Sigma-Aldrich; 19–148) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM and cells were treated at a concentration of 100 nM for the times indicated.
ATG5 siRNA (human [Santa Cruz Biotechnology; sc-41445]), IFNAR siRNA (human [Santa Cruz Biotechnology; sc-35637]), GABARAPL1 siRNA (human [Santa Cruz Biotechnology; sc-105386]), GABARAPL2 siRNA (human [Santa Cruz Biotechnology; sc-41958]), and TBK1 siRNA (human [Santa Cruz Biotechnology; sc-39058]) were reconstituted by following the manufacturer-provided datasheet. HeLa cells were transfected using Effectene transfection reagent by following the manufacturer’s guidelines for reagent volumes. Forty-eight hours following transfection, the medium was refreshed and cells were subsequently infected.

Sawaged S., Mota T., Piplani H., Thakur R., Lall D., McCabe E., Seo S., Sutterwala F.S., Feuer R., Gottlieb R.A, & Sin J. (2022). TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles. PLoS Pathogens, 18(8), e1010350.

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siRNA-mediated LY6D and ATG5 knockdown

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ON-TARGETplus Smart Pool siRNA for LY6D (L-012615-01) and its control siRNA (D-001810-10) were from GE Healthcare Dharmacon (Lafayette, CO). ATG5 siRNA (sc-41445) was obtained from Santa Cruz Biotechnology. Cells were seeded and transfected with 30-nM siRNA using HiPerFect Transfection Reagent (Qiagen, Venlo, Netherlands) according to the manufacturer’s instruction.

Nagano T., Iwasaki T., Onishi K., Awai Y., Terachi A., Kuwaba S., Asano S., Katasho R., Nagai K., Nakashima A., Kikkawa U, & Kamada S. (2020). LY6D-induced macropinocytosis as a survival mechanism of senescent cells. The Journal of Biological Chemistry, 296, 100049.

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Culturing and Analyzing HUVEC Cells

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Human umbilical vein endothelial cells (HUVEC) were grown and maintained in basal media supplemented with VascuLife EnGS LifeFactors Kit (Lifeline Cell Technology, Oceanside, CA). Cells were plated on 0.2% (w/v) gelatin-coated cell culture plates (Thermo Fisher Scientific, Waltham, MA) and utilized within the first five passages. Antibodies for Western Blotting (WB) were purchased from the following sources and utilized at the designated dilutions: GAPDH (14C10, 1:10,000 dilution; Cell Signaling, Danvers, MA), LC3B (L7543, 1:1,000 dilution; Sigma), and HAS2 (sc-514737 and sc-365263 at 1:250 and 1:150, respectively; Santa Cruz Biotechnology, Dallas, TX). The following siRNA was purchased from Santa Cruz Biotechnology and used at the designated amounts: ATG5 siRNA (sc-41445, 100 pM) and scramble control siRNA-A (sc-37007, 100 pM).

Lüscher M., Hochstrasser U., Vogel G., Aeschbacher R., Galati V., Nelson C.J., Boller T, & Wiemken A. (2000). Cloning and Functional Analysis of Sucrose:Sucrose 1-Fructosyltransferase from Tall Fescue. Plant Physiology, 124(3), 1217-1228.

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