The NF-κB and p53-dependent luciferase reporter plasmids, the constructs expression FLAG-IKKα and FLAG-IKKα-KM were described in our previous reports14 (link),17 (link). The following primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): Beclin-1, LC3B, SQSTM1, ATG5, phospho-p53-Ser15, p53, phospho-IKKα-Ser176/180, IKKα, IKKβ, IκBα, phospho-CHK1-Ser345, CHK1, FLAG, and ACTB. The following primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): IKKγ, GADD45α, and DRAM1. IKKα siRNA, p53 siRNA, and ATG5 siRNA were purchased from Cell Signaling Technology (Beverly, MA, USA). DRAM1 siRNA and CHK1 siRNA were purchased from Riobo Technology (Guangzhou, China). 3-MA, BafA1, and MG132 were ordered from Sigma-Aldrich (St. Louis, MO, USA).
Atg5 sirna
Manufactured by Cell Signaling Technology
Sourced in United States
ATG5 siRNA is a synthetic small interfering RNA (siRNA) that targets the ATG5 gene, which is involved in the autophagy process. This product is designed to modulate the expression of the ATG5 gene, which plays a crucial role in the formation of autophagosomes during autophagy. The siRNA can be used as a research tool to study the function of ATG5 and its involvement in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Sqstm1, by Cell Signaling Technology (1 mentions)
Bafa1, by Merck Group (1 mentions)
Phospho ikkα ser 176 180, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Phospho chk1 ser345, by Cell Signaling Technology (1 mentions)
Gadd45α, by Santa Cruz Biotechnology (1 mentions)
Dram1, by Santa Cruz Biotechnology (1 mentions)
P53 sirna, by Cell Signaling Technology (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Mg132, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Ab8224, by Abcam (1 mentions)
Lc3b sirna, by Cell Signaling Technology (1 mentions)
Penicillin, by ScienCell (1 mentions)
Huvecs, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by ScienCell (1 mentions)
Huvecs, by ScienCell (1 mentions)
6 protocols using atg5 sirna
1
Investigating NF-κB and p53 Signaling
2
Autophagy Regulation in Colorectal Cancer
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SW480 and SW620 were obtained from American Type Cell Collection (ATCC/LGC promochem, Molsheim, France), authenticated by the seller and cultured as previously described 7 in RPMI 1640 medium (Gibco Life Technologies, Paisley, UK). SW480 was a primary CRC‐derived cell line (i.e. stage II), whereas SW620 was derived from the lymph node (i.e. stage III) of the same patient. Cells were used when they reached 80% subconfluence. Cell cultures were used for a maximum of 10 passages.
INTERFERIN transfection reagent (Polyplus; Cell Signaling Technology) was used for siRNA transfection. In total, 200,000 cells per well were transfected in 6‐well plates with either 20 nM of negative control or ATG5 siRNA (Cell Signaling Technology). After 72 hrs with fresh medium, the cells were washed and recovered as described before. Silencing was confirmed by Western blotting.
INTERFERIN transfection reagent (Polyplus; Cell Signaling Technology) was used for siRNA transfection. In total, 200,000 cells per well were transfected in 6‐well plates with either 20 nM of negative control or ATG5 siRNA (Cell Signaling Technology). After 72 hrs with fresh medium, the cells were washed and recovered as described before. Silencing was confirmed by Western blotting.
3
Knockdown of LC3B and Atg5 in HeLa Cells
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HeLa cells were transfected with LC3B siRNA (Cell Signalling Technology, 6213, 100 nM) or Atg5 siRNA (Cell Signalling Technology, 6345, 100 nM) using Lipofectamine RNAiMAX as per manufacturer protocol. The efficiency of knockdown was assessed at 24 h (for LC3) and 48 h (for Atg5) after transfection by western blot. The following primary antibodies were used: LC3 (rabbit polyclonal, MBL, PM036, 1:1000), Atg5 (rabbit polyclonal, Cell Signalling Technology, 2630, 1:1000) and β actin (mouse monoclonal, Abcam, ab8224, 1:1000). The expression level of the target genes was calculated as the ratio of target gene to β actin (loading control).
4
Endothelial Cell Autophagy Regulation
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HRGECs and HUVECs were obtained from ScienCell Research Laboratories (4000, 8000, ScienCell; Santiago, USA) and cultured with endothelial cell medium (1001, ScienCell; Santiago, USA), containing 10% FBS (0025, ScienCell; Santiago, USA), 5 ml endothelial cell growth supplement (1052, ScienCell; Santiago, USA) and 5 ml penicillin (10,000 U/ml)/streptomycin (10,000 µg/ml) solution (0503, ScienCell; Santiago, USA). Cells were incubated with IL-6 at indicated time points or different concentrations of IL-6 for 24hours. 3-MA (10 mM) was used as an autophagy inhibitor in vitro experiment.
For transfection, HUVECs that were cultured in 6-well plates with non-proliferative medium. The HUVECs were transfected with 25nM of ATG5 siRNA (6345, Cell Signaling Technology; Boston, USA) or control siRNA using Lipofectamine 2000 transfection reagent (11668–019, Invitrogen; California, USA) according to the manufacturer’s instructions.
For transfection, HUVECs that were cultured in 6-well plates with non-proliferative medium. The HUVECs were transfected with 25nM of ATG5 siRNA (6345, Cell Signaling Technology; Boston, USA) or control siRNA using Lipofectamine 2000 transfection reagent (11668–019, Invitrogen; California, USA) according to the manufacturer’s instructions.
5
Atg5 Silencing in Human Endothelial Cells
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Atg5 siRNA (#6345) was purchased from Cell Signaling Technology. Control siRNA (AM4611) was purchased from Thermo Fisher Scientific. siRNA was transfected into cells with Lipofectamine RNAiMAX kit (Thermo Fisher Scientific) following manufacturer’s instructions. In brief, HUVECs were grown in 0.1% gelatin coated 6-well plate to 80% confluence. siRNA was diluted in Opti-MEM medium and mixed with Lipofectamine RNAiMAX reagent for 5 min at room temperature before transfection. Cells were transfected with the mixture containing 25 pmol siRNA per well and incubated for 72 h.
6
Autophagy Regulation in LPS-Treated A549 Cells
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Cells were transiently transfected with an enhanced green fluorescent protein (EGFP)-LC3 plasmid (constructed by GeneChem Co. Ltd, Shanghai, China) using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer's instructions. After a 48-h transfection, the cells were treated with LPS at 50 µg/ml for another 16 h and then subjected to laser confocal scanning microscopy (TCS SP5; Leica Microsystems, Mannheim, Germany).
For the RNA interference experiments, A549 cells were transfected with Beclin-1 short interfering RNA (siRNA), Atg5 siRNA (Cell Signaling), PERK siRNA, ATF4 siRNA (Santa Cruz), or control siRNA or co-transfected with EGFP-LC3 plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen) as directed by the manufacturer. After 48 h, the effect of the siRNA treatment was assessed via Western blotting analysis. In parallel, cells were treated with LPS and analysed as described above.
For the RNA interference experiments, A549 cells were transfected with Beclin-1 short interfering RNA (siRNA), Atg5 siRNA (Cell Signaling), PERK siRNA, ATF4 siRNA (Santa Cruz), or control siRNA or co-transfected with EGFP-LC3 plasmid using Lipofectamine 3000 Transfection Reagent (Invitrogen) as directed by the manufacturer. After 48 h, the effect of the siRNA treatment was assessed via Western blotting analysis. In parallel, cells were treated with LPS and analysed as described above.
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