Hif 1α sirna

Manufactured by RiboBio

Sourced in China

HIF-1α siRNA is a laboratory tool used to study the expression and function of the hypoxia-inducible factor-1 alpha (HIF-1α) gene. It is a small interfering RNA (siRNA) designed to target and silence the HIF-1α gene, which plays a key role in the cellular response to low oxygen conditions.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Human serum albumin (hsa), by Beyotime (1 mentions) Transwell co culture system, by Corning (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Mcherry gfp lc3, by Addgene (1 mentions) Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Fluorescence microscope, by Leica (1 mentions) Gfp lc3, by Addgene (1 mentions) Cobalt chloride, by Merck Group (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Hif 1α sirna, by Thermo Fisher Scientific (1 mentions) Beclin1 sirna, by RiboBio (1 mentions)

8 protocols using hif 1α sirna

Modulation of Cellular Interactions via Rab27a and HIF-1α

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The human tubular epithelial cell line HK-2 and the human myeloid leukemia mononuclear cell line THP-1 were obtained from the Cell Bank of the Type Culture Collection (Chinese Academy of Sciences, Shanghai, China) and maintained in Roswell Park Memorial Institute 1640 medium containing 10% fetal bovine serum (FBS) (Gibco, Australia) at 37 °C under a 5% CO₂ atmosphere.
For treatment with human serum albumin (HSA), HK-2 cells were cultured in RPMI 1640 medium containing 2% FBS for 24 h and then stimulated with 20 mg/ml HSA for 48 h. For lipopolysaccharide (LPS) stimulation, THP-1 cells were induced to macrophages using 100 nM phorbol myristate acetate (PMA) (S1819; Beyotime, China) for 48 h and treated with 100 ng/ml LPS (S1732; Beyotime) for another 24 h. For investigations with the prolyl hydroxylase inhibitor FG-4592, macrophages were treated with 5 mM FG-4592 (SC1135; Beyotime) for 24 h.
HK-2 cells and macrophages were transfected using Lipofectamine 3000 (Invitrogen, Carlsbad, USA). To knock down Rab27a, HK-2 cells were transfected with 50 nM Rab27a siRNA (RiboBio, Guangzhou, China). For HIF-1α knockdown, macrophages were transfected with 50 nM HIF-1α siRNA (RiboBio, Guangzhou, China). Transwell co-culture systems were applied for co-culture with HK-2 cells and macrophages (Corning, MA, USA).

Jia Y., Chen J., Zheng Z., Tao Y., Zhang S., Zou M., Yang Y., Xue M., Hu F., Li Y., Zhang Q., Xue Y, & Zheng Z. (2022). Tubular epithelial cell-derived extracellular vesicles induce macrophage glycolysis by stabilizing HIF-1α in diabetic kidney disease. Molecular Medicine, 28, 95.

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Studying Autophagy Using Transfection

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Transfection was achieved using Lipofectamine 2000 Transfection Reagent (Invitrogen, 11668-019) following the manufacturer’s protocol. Cells were transfected with plasmids encoding mCherry-GFP-LC3 (22418) and GFP-LC3 (22405) from Addgene, Cambridge, MA, USA. Human pcDNA3.1(+)-NDRG1 plasmid constructed by Gene-Chem Co. Ltd (Shanghai, China). Human NDRG1 siRNA, HIF-1α siRNA and nontarget siRNA were purchased from Ribobio (Guangzhou, China). Cells were cultured in six-well plates and transfected with plasmids for 24 h. After the designated treatments, live cell images were obtained using a fluorescence microscope (Leica, Wetzlar, Germany). Protein knockdown or overexpression efficiency was assessed by immunoblotting.

Wang H., Li W., Xu J., Zhang T., Zuo D., Zhou Z., Lin B., Wang G., Wang Z., Sun W., Sun M., Chang S., Cai Z, & Hua Y. (2017). NDRG1 inhibition sensitizes osteosarcoma cells to combretastatin A-4 through targeting autophagy. Cell Death & Disease, 8(9), e3048-.

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Cobalt-Induced Chondrocyte Modulation

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Chondrocyte culture medium was supplemented with different concentrations of cobalt chloride (0, 50, 10, 150 and 200 µM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for the indicated times. When the growth plate chondrocytes had reached ~50% confluence, the original culture medium was discarded, and OPTIMEM medium containing YAP small interfering (si)RNA (50 nM), HIF-1α siRNA (50 nM; Guangzhou Ribobio Co., Ltd., Guangzhou, Guangdong, China) or negative control (NC) siRNA, and Lipofectamine 2000^®(Invitrogen; Thermo Fisher Scientific, Inc.), was transfected in chondrocytes for 6 h. The sequences of YAP siRNA were as follows: GCCATGAACCAGAGGATCA. The sequences of HIF-1α siRNA were as follows: CTGATAACGTGAACAAATA, TCGACAAGCTTAAGAAAGA and GGACAATATAGACATT. Subsequent experiments were performed after 24 h. Endogenous YAP and HIF-1a protein knock-out efficiency was examined by western blot analysis.

Li H., Li X., Jing X., Li M., Ren Y., Chen J., Yang C., Wu H, & Guo F. (2018). Hypoxia promotes maintenance of the chondrogenic phenotype in rat growth plate chondrocytes through the HIF-1α/YAP signaling pathway. International Journal of Molecular Medicine, 42(6), 3181-3192.

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HIF-1α Downregulation in HUVECs

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To downregulate the expression of HIF-1α in HUVECs, we used siRNA for cell transfection, and Lipofectamine 2000 was used to improve the interference efficiency. In detail, we plated cells to 70% confluence at the time of transfection. Then, Lipofectamine 2000 Reagent (Invitrogen, California, USA) or HIF-1α siRNA (5'-GGAACATGATGGTTCACTT-3', RiboBio, Guangdong, China) was premixed and incubated with Opti-MEM (Gibco, MA, USA) for 10 min and incubated with both mixtures for 5 min (in 6-well plates, 5 μL of Lipofectamine 2000 Reagent, 5 μL of HIF-1α siRNA and 300 μL of Opti-MEM in each well). The medium on the plate was removed and washed with PBS twice. Finally, medium with no serum and a siRNA mixture were added for incubation. The medium was removed 6 h after transfection and replaced with fresh medium containing 10% serum.

Wang R., Zhang Z., Xu Z., Wang N., Yang D., Liu Z.Z., Liao Q., Xia X., Chen C., Shou J., Li L., Wang W.E., Zeng C., Xia T, & Wang H. (2021). Gastrin mediates cardioprotection through angiogenesis after myocardial infarction by activating the HIF-1α/VEGF signalling pathway. Scientific Reports, 11, 15836.

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HIF-1α Knockdown in HRCECs

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Regarding as HIF-1α knockdown experiment, HRCECs were transfected with HIF-1α siRNA (10 nM) for 24 h using Lipofectamine 3000 reagent (Invitrogen). The sequence of HIF-1α siRNA: sense, 5’-CAGAAAUGGCCUUGUGAAA-3’; antisense, 5’-UUUCACAAGGCCAUUUCUG-3’. HIF-1α siRNA was provided with RiboBio.

Kong L., Li J., Yang Y., Tang H, & Zou H. (2022). Paeoniflorin alleviates the progression of retinal vein occlusion via inhibiting hypoxia inducible factor-1α/vascular endothelial growth factor/STAT3 pathway. Bioengineered, 13(5), 13622-13631.

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Hypoxia-Induced Autophagy via HIF-1α

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Cells were exposed to the HIF-1α inhibitor 2ME2 to determine the mechanisms of hypoxia-induced autophagy. BV2 cells were seeded in six-well plates and pretreated with 3 µM 2ME2 for 6 h prior to hypoxia exposure. These pretreated BV2 cells were exposed to hypoxia for 3, 6, 9, 12 and 24 h. Supernatants from cell cultures were collected and assayed for secreted cytokines, and cell pellets were used for RNA isolation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. For transfection with siRNA, cells were plated in six-well plates at 5×10⁵ cells/well. Plated cells were grown in DMEM supplemented with 10% FBS overnight, and transfected with 50 nM HIF-1α siRNA (5′-CTACTCAGGACACAGATTTAGACTTGGAG-3) or negative control siRNA (5′-CAGCAACCAGGTGACCGTG-3′) obtained from Guangzhou Ribobio Co. Ltd., Guangzhou, China, using the transfection reagent Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific Inc.) according to the manufacturer's protocol, 24 h prior to treatment with drugs.

Wang X., Ma J., Fu Q., Zhu L., Zhang Z., Zhang F., Lu N, & Chen A. (2017). Role of hypoxia-inducible factor-1α in autophagic cell death in microglial cells induced by hypoxia. Molecular Medicine Reports, 15(4), 2097-2105.

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HIF-1α Overexpression and Silencing

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HIF-1α-overexpressing pcDNA 3.0 plasmid (Invitrogen) and HIF-1α siRNA (RiboBio Guangzhou, China) were generated and transfected into AGS and MKN45 cells at a final concentration of 2 ng and 10 nM, respectively. The process of transfection was conducted by using Lipofectamine 3000 reagent (Invitrogen) as per the standard protocols.

Liu J., Liu H., Zeng Q., Xu P., Liu M, & Yang N. (2020). Circular RNA circ-MAT2B facilitates glycolysis and growth of gastric cancer through regulating the miR-515-5p/HIF-1α axis. Cancer Cell International, 20, 171.

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Transfecting Human Endometrial Cells

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Human lncRNA‐MALAT1 small interfering RNA (siRNA), HIF‐1α siRNA, Beclin1 siRNA, and non‐specific negative control siRNA (si‐NC) were synthesized by RIBOBIO (Guangzhou, Guangdong, China). HIF‐1α overexpression plasmid (pG/CMV/HIF‐1α/IRES/EGFP) and empty vector plasmid were purchased from Gemma Pharmaceutical Technology (Suzhou, Jiangsu, China). Briefly, human endometrial stromal cells (2 x 10⁵ cells/well) were seeded in 6‐well plates and grown to 60%‐80% confluence, and then transfected with 50 n mol L⁻¹ siRNA using transfection reagent Lipofectamine 2000 (Invitrogen Life Technologies, USA) according to the manufacturer's instructions. After a 6 hours antibiotic‐free medium incubation, the transfection mixture was removed, and the cells were incubated in DMEM/F‐12 with 20% FBS for another 24 hours, followed by further drug treatments. Sequences for siRNAs used in this study are shown in Table S2.

Liu H., Zhang Z., Xiong W., Zhang L., Du Y., Liu Y, & Xiong X. (2018). Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis. Journal of Cellular and Molecular Medicine, 23(1), 439-452.

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