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. 2021 Oct 9;12(10):924. doi: 10.1038/s41419-021-04234-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic representation of the guide RNA targeting NME2 gene. The sequence of PAM (protospacer adjacent motif) was boxed. b T7 endonuclease 1(T7E1) assay. Gastric cancer stem-like cells (MKN-45) were transfected with pHBCas9/gRNA-Pure plasmid containing NME2 gRNA. As a control, vector only (without NME2 gRNA) was included in the transfection. The DNA containing target site of NME2 gRNA was amplified by PCR using the genomic DNA extracted from the transfected cells. The PCR product was digested with T7E1, followed by agarose gel electrophoresis. M, DNA marker. c Sequencing analysis of NME2 in wild-type (WT) and NME2-knocked out (KO) gastric cancer stem-like cells. The sequences of two alleles of NME2 mutant were indicated. PAM was boxed. d Western blot analysis of NME2 in NME2-knockout (NME2 KO) gastric cancer stem-like cells. β-Tubulin was used as a control. e Impact of NME2 knockout and rescue on the cell number of gastric cancer stem-like cells. The cells transfected with vector alone or pcDNA-NME2 plasmid were cultured and counted at 1st, 2nd, and 3rd days (^**p < 0.01). f Influence of NME2 knockout and rescue on the cell viability of gastric cancer stem-like cells. The cells transfected with vector alone or pcDNA-NME2 plasmid were cultured for different times, followed by the examination of cell viability (^**p < 0.01). g Impact of NME2 knockout and rescue on cell cycle of gastric cancer stem-like cells. The gastric cancer stem-like cells containing WT, KO, or rescue NME2 were cultured for 48 h and then subjected to cell cycle examination with flow cytometry (^**p < 0.01). h Effects of NME2 knockout or rescue on apoptosis of gastric cancer stem-like cells. The cells with different treatments were cultured for 48 h. Subsequently, the caspase 3/7 activity of the cells was examined (^**p < 0.01). i Detection of apoptosis using Annexin V (^**p < 0.01). j Tumorsphere formation assay of the NME2 -knocked out or -rescued gastric cancer stem-like cells. At day 8 after the inoculation of a single cell, the percentage of tumorsphere formation was calculated (^**p < 0.01). k Effects of NME2 knockout and rescue on the expressions of stemness genes in gastric cancer stem-like cells. The cells with different treatments were cultured for 48 h, followed by quantitative real-time PCR and western blot (^**p < 0.01).