Knockdown of ASH1L methyltransferase induced apoptosis inhibiting proliferation and H3K36 methylation in bovine cumulus cells
Lixin CuiYa-Qing TianHaisheng HaoHuiying ZouYunwei PangShanjiang ZhaoXueming ZhaoHuabin ZhuWeihua Du
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Objective To quantify the early apoptosis of gastric cancer cell line SCG7901 induced by 5 FU flow cytometry with Annexin V FITC and PI double labeling. Methods The tumor cells were treated with 5 FU at concentration of 250, 125, 50, and 25 mg/L. The apoptosis was detected 0, 6, 12, 18, 24, 30 h after the treatment by using flow cytometry with Annexin V FITC and PI double labeling method. Results Early apoptosis percentage of tumor cells (0-24 h) was increased significantly with the affected time and the content of 5 FU. But it was decreased during 24-30 h after the treatment, while more necrotic cells were seen at same time. Conclusion 5 FU can induce early apoptosis of tumor cells in a time and dosage depended manner. Annexin V FITC/PI double labeling flow cytometry can distinguish dead cells from early apoptotic cells and be of great use in tumor cell apoptosis quantitative analysis.
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Annexin A5
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目的 用一种新的荧光染色技术(FLICA/PI)检测放射诱导T淋巴细胞白血病细胞(MOLT-4)的细胞凋亡过程中胱氡肽酶 3(Caspase 3)原位激活及探讨其活性变化特点,并与亚G1峰和膜联蛋白 V/碘化丙啶(Annexin V/PI)检测细胞凋亡方法的灵敏度进行比较性评价.方法以放射诱导的MOLT-4细胞凋亡为模型,用荧光标记的Caspase 3抑制剂(FLICA)标记原位激活的Caspase 3,然后,用流式细胞仪定量检测并分析其活性变化;同时,用亚G1峰法和Annexin V/PI检测细胞凋亡.结果 FLICA/PI检测到的Caspase 3激活,在照射后2 h开始升高(5.84%),而Annexin V/PI在4 h时检测到的凋亡率为5.35%,FLICA/PI比Annexin V/PI可提前2 h检测到细胞凋亡,Caspase 3活性的出现早于细胞膜反转.FLICA阳性率在照射后4 h暴发性增高至12.39%,显示有时间效应.两种检测方法均比亚G1峰法敏感, 但Annexin V/PI检测的凋亡率低于FLICA/PI法.结论 X-射线能诱导MOLT-4细胞凋亡,并有明显的时间效应关系;Caspase 3激活有阈值效应,是照射后细胞发生凋亡过程中早于形态学变化的检测指标.用Caspase 3活性检测细胞凋亡更直接,更敏感。
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RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction.Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP.Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.
Knockdown resistance
RNA Silencing
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[Objective]The research aimed compare the advantages and disadvantages of four methods for apoptosis assay. [Method]Flow cytometry was used to examine cell apoptosis which labeled with PI,Annexin V-FITC /PI,Rhodamine 123 and Fluo-3 respectively. [Result]Each menthod had its merits and drawbacks,and multi-parameter analysis should be considered in the inspection process. [Conclusion]It could provide experimental support for sequential studies.
Rhodamine 123
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To develop a method of analyzing apoptosis using multi-parameter flow cytometry, HL60 cell line was incubated with Annexin V-FITC/PI after exposure to etoposide, and the apoptosis of HL60 cell was monitored by flow cytometry. The result showed that the percentage of apoptosis cell increases with the time of exposure to etoposide. In conclusion, Annexin V FITC/PI method can not only detect special proteins, but also monitor the integrity of cell membrane. Multi-parameter flow cytometry is a rapid, easy and accurate method for the detection of apoptosis.
Cytometry
Annexin A5
HL60
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Objective The current study was designed to compare the sensitivities of Calcein-AM and Annexin V-FITC for early detection of apoptosis in living cells by flow cytometry and to establish a new method for early detection of apoptosis. Methods The Molt-4 cell line treated with camptothecin (CPT) or ultraviolet(UV) were labeled with Calcein-AM and FITC-conjugated Annexin V respectively, then the apoptosis rate were analyzed by flow cytometry and the performance of Calcein-AM and Annexin V for early detection of apoptosis were compared. Results Our results showed that both probes allowed the detection of apoptotic cells. However, Calcein-AM was more sensitive than Annexin V. We could detect the apoptosis induced by CPT in two hours after inducing with Calcein-AM and in three hours after inducing with Annexin V. The apoptosis induced by UV could be detected in one hours after inducing with Calcein-AM but two hours after inducing with Annexin V. Conclusion Annexin V is less sensitive than Calcein-AM for detection of early apoptosis, and the new method for detection of early apoptosis with Calcein-Am is stable, reliable, sensitive and low-cost.
Calcein
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To evaluate the method of measuring apoptosis by flow cytometry with three stains, the apoptosis percentage of rat cortical neurons was measured by flow cytometry using propidium iodide(PI) staining, Annexin V/PI and JC-1 double staining respectively. The results showed that: the apoptotic positive percentage measured by PI staining,Annexin V/PI and JC-1 was 2.60%,6.52% and 18.56%, respectively. Our conclusion is that there were significant differences among the results of three staining methods: JC-1 staining was the most sensitive method, whereas PI single staining was not suitable to measure the early apoptosis of cells.
Propidium iodide
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Annexin A5
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Objective To evaluate the application of flow cytometry with annexin V FITC / PI double staining for detecting the curcumin-induced apoptosis of hepatic stellate cells(HSC).Methods The HSC line T6 was incubated with curcumin at different concentrations for 24 hours.Apoptosis of the cells was detected by flow cytometry with annexin V FITC / PI double staining.Results The percentage of the apoptotic cells increased after the treatment with 10 μmol/L of curcumin and that of the necrotic cells elevated under the condition of 30 μmol/L.The apoptotic rate was(3.81 ± 0.64)% in the group with 0 μmol / L,(6.52 ± 1.88)% in the group with 10 μmol / L,(11.61 ± 2.79)% in the group with 20 μmol / L,(52.03 ± 4.38)% in the group with 30μmol / L,and(87.11 ± 12.62)% in the group with 40μmol / L,and it was significantly higher in the latter three groups than in the group with 0 μmol / L(P 0.05).Conclusion Flow cytometry with annexin V FITC / PI double staining is a quantitative method for determinating the early apoptosis of HSC induced by curcumin.
Hepatic stellate cell
Cytometry
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We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.
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