A Pitfall of Propidium Iodide Staining in Fluorescence-Activated Cell Sorting Cell Cycle Analysis?

Buchegger, Franz; Dupertuis, Y.M.; Perillo-Adamer, Florence

doi:10.1158/0008-5472.can-06-3663

Cited by 5 publications

(3 citation statements)

References 3 publications

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“…Flow cytometric analysis. The cell apoptosis rate was determined by f low cytometric analysis with annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining (25,26). Briefly, 1x10 5 lung cancer cells were plated into 6-well plates.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of microRNA-125a-3p effectively inhibits the cell growth and invasion of lung cancer cells by regulating the mouse double minute 2 homolog/p53 signaling pathway

Li¹,

Li²,

Zhao³

et al. 2015

Molecular Medicine Reports

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MicroRNAs (miRs) are a family of small non-coding RNAs that are 21‑24 nucleotides in length. Decreased expression of hsa‑miR‑125a‑3p is observed in a number of patients with non‑small cell lung cancer; however, it is not clear how this miRNA regulates the growth and invasion of lung tumor cells. The aim of the present study was to identify the function of hsa‑miR‑125a‑3p in the growth and invasion of lung cancer cells. The expression of hsa‑miR‑125a‑3p in the A549, NCI‑H460 and SPCA‑1 lung cancer cell lines was analyzed by reverse transcription‑quantitative polymerase chain reaction and the human bronchiolar epithelium cell line (HBE) was used as a control. The results demonstrated that the expression of hsa‑miR‑125a‑3p was significantly lower in NCI‑H460, A549 and SPCA‑1 cells, compared with that in HBE cells. Overexpression of sense miR‑125a‑3p in the A549 lung cancer cell line inhibited cell proliferation for 5‑7 days (P<0.01), and transfection of antisense miR‑125a‑3p did not suppress the cell growth of the lung cancer cells. In addition, overexpression of miR‑125a‑3p in the NCI‑H460 lung cancer cell line markedly induced cell apoptosis, which was detected by fluorescence‑activated cell sorting with annexin V‑fluorescein isothiocyanate/propidium iodide staining. The results of the Transwell migration assay also revealed that transfection of miR‑125a‑3p resulted in decreased migration of lung cancer tumor cells. The pro‑apoptotic gene p53 expression was detected by western blot analysis. The results revealed that the expression of mouse double minute (MDM)‑2 homolog, the principal cellular antagonist of p53, was decreased and p53 expression was upregulated in sense has‑miR‑125a‑3p transfected A549 cells. This was consistent with that observed in NCI‑H460 cells, suggesting that hsa‑miR‑125a‑3p may be involved in the regulation of the MDM2/p53 signaling pathway in lung cancer cells. In conclusion, overexpression of hsa‑miR‑125a‑3p significantly inhibited the proliferation and invasion of lung cancer cells, which may aid in determining the mechanisms underlying the development of lung cancer.

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Section: Methodsmentioning

confidence: 99%

Overexpression of microRNA-125a-3p effectively inhibits the cell growth and invasion of lung cancer cells by regulating the mouse double minute 2 homolog/p53 signaling pathway

Li¹,

Li²,

Zhao³

et al. 2015

Molecular Medicine Reports

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show abstract

“…Since the amount of stain in the cells at each phase is a continuum rather than a fixed value, differentiating the number of cells in the border areas between G 0 /G 1 and S or S and G 2 /M is not so easy. 51 This problem could be partially overcome by adding labeled nucleotides to the medium during the cell culture and performing a dual color analysis in addition to the simple staining by PI. This latter approach will be further addressed later.…”

Section: Flow Cytometrymentioning

confidence: 99%

Effect of Nanoparticles on the Cell Life Cycle

et al. 2011

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“…They speculated that this premature degradation confers resistance to FdUrd by preventing the futile BER response. However, there has been a question regarding the interpretation of the FACS analysis used to conclude that the proposed premature UNG2 degradation actually occurred in S-phase [72]. Nonetheless, Fischer et al showed at least in HeLa cells that siRNA against UNG2 conferred resistance to FdUrd [71], offering evidence in support of the futile cycling model.…”

Section: Base Excision Repair and 5-fu Resistancementioning

confidence: 99%

Participation of DNA repair in the response to 5-fluorouracil

Wyatt

Wilson

2008

Cell. Mol. Life Sci.

215

207

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The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anticancer treatments.

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A Pitfall of Propidium Iodide Staining in Fluorescence-Activated Cell Sorting Cell Cycle Analysis?

Cited by 5 publications

References 3 publications

Overexpression of microRNA-125a-3p effectively inhibits the cell growth and invasion of lung cancer cells by regulating the mouse double minute 2 homolog/p53 signaling pathway

Overexpression of microRNA-125a-3p effectively inhibits the cell growth and invasion of lung cancer cells by regulating the mouse double minute 2 homolog/p53 signaling pathway

Effect of Nanoparticles on the Cell Life Cycle

Participation of DNA repair in the response to 5-fluorouracil

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