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Effects of hypoxia on lung cancer cells

ABSTRACT: 300,000 of H358 cells were seeded in 6-well plates in quadruplicates per plate and placed either at 1 % or 21 % oxygen for either 48 or 72h, respectively. Upon the indicated time, cells were harvested in 500 µl of harvesting solution (80 % methanol in 50 mM ammonium acetate, supplemented with 2.5 mM N-ethylmaleimide (NEM) and heavy glutathione internal standard), sonicated for 5 s at 10 % amplitude and processed by for one-pot redox metabolite and protein thiol analysis approach. This project is in vitro validation of findings connected to the PRIDE project PXD052340.

INSTRUMENT(S): timsTOF HT

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Lung

SUBMITTER: Tamara Tomin

LAB HEAD: Ruth Birner-Gruenberger

PROVIDER: PXD062503 | Pride | 2025-05-12

REPOSITORIES: Pride

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			Action	DRS
	20230316_HumanCr_20806.fasta	Fasta
	DIANNoutput.zip	Other
	RawDataDIA.zip	Other
	checksum.txt	Txt

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GAPDH inhibition in A549 and H358 lung cancer cells

Project description:Human lung cancer cell lines A549 and H358 were obtained from (Biobank of Medical University of Graz) and cultured in RPMI 1640 (R0883; Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco), 5 mM glutamine and 5 mM Pen Step, and sub-cultured regularly. For siRNA mediated knock-down of GAPDH, 150,000 A549 and H358 cells were seeded in triplicates of a six-well plate and transfected upon reaching ca. 60% confluency with silencerTM siRNA kit targeting GAPDH or non-target control (AM4605; Thermo), using RNAiMax (Thermo) and according to the manufacturer’s instructions for 6-well plate scale. For GAPDH inhibition, 150.000 A549 and H358 cells were seeded in six replicates per condition, then treated with 10 µM koningic acid (KA) or vehicle control (dmso). 48 h post transfection or 24 h post KA treatment, cells were harvested in 500 µl of harvesting solution (80% methanol in 50 mM ammonium acetate with 2.5 mM N-ethylmaleimide) and processed according to one-pot redox approach.

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