Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3

2016 ◽  
pp. 313-329 ◽  
Author(s):  
Sandra Muñoz-Braceras ◽  
Ricardo Escalante
Keyword(s):  
Hela Cells ◽  
Autophagic Flux

Gadolinium Oxide Nanoparticles Enhance the Cytotoxicity of Chemotherapeutic Drugs by Blocking Autophagic Flux in Human Ovarian Cancer Cells

2020 ◽  
Vol 16 ◽  
Author(s):  
Zhixiong Xie ◽  
Tianyu Zhang ◽  
Cheng Zhong
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Hela Cells ◽  
Late Stage ◽  
Ovarian Cancer Cells ◽  
Oxide Nanoparticles ◽  
Autophagic Flux ◽  
Human Ovarian Cancer ◽  
Chemotherapeutic Drugs ◽  
Chemotherapy Drugs

Background: During chemotherapy, drugs can damage cancer cells’ DNA and cytomembrane structure, and then induce cell death. However, autophagy can increase the chemotherapy resistance of cancer cells, reducing the effect of chemotherapy. Objective: To block the autophagic flux in cancer cells, it is vital to enhance the anti-cancer efficacy of chemotherapy drugs; for this purpose, we test the gadolinium oxide nanoparticles (Gd2O3 NPs)’ effect on autophagy. Methods: The cytotoxicity of Gd2O3 NPs on HeLa cells was evaluated by a (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then, monodasylcadaverine staining, immunofluorescence, immunoblot and apoptosis assay were conducted to evaluate the effect of Gd2O3 NPs on autophagy and efficacy of chemotherapy drugs in human ovarian cancer cells. Results: We found that Gd2O3 NPs, which have great potential for use as a contrast agent in magnetic resonance imaging, could block the late stage of autophagic flux in a dose-dependent manner and then cause autophagosome accumulation in HeLa cells. When co-treated with 8 μg/mL Gd2O3 NPs and 5 μg/mL cisplatin, the number of dead HeLa cells increased by about 20% compared with cisplatin alone. We observed the same phenomenon in cisplatin-resistant COC1/DDP cells. Conclusion: We conclude that Gd2O3 NPs can block the late stage of autophagic flux and enhance the cytotoxicity of chemotherapeutic drugs in human ovarian cancer cells. Thus, the nanoparticles have significant potential for use in both diagnosis and therapy of solid tumor.

scholarly journals Inhibition of autophagic flux by ROS promotes apoptosis during DTT-induced ER/oxidative stress in HeLa cells

2016 ◽  
Vol 35 (6) ◽  
pp. 3471-3479 ◽  
Author(s):  
XI-YAN XIANG ◽  
XIAO-CHUN YANG ◽  
JIN SU ◽  
JING-SONG KANG ◽  
YAO WU ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Hela Cells ◽  
Autophagic Flux

scholarly journals Salmonella spvC Gene Inhibits Autophagy of Host Cells and Suppresses NLRP3 as Well as NLRC4

2021 ◽  
Vol 12 ◽  
Author(s):  
Liting Zhou ◽  
Yuanyuan Li ◽  
Song Gao ◽  
Haibo Yuan ◽  
Lingli Zuo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Laser Scanning ◽  
Systemic Infection ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Laser ◽  
Autophagic Flux ◽  
Virulence Determinants ◽  
Pyrin Domain ◽  
Lyase Activity

Salmonella spvC gene, encoding a phosphothreonine lyase on host mitogen-activated protein kinases, facilitates systemic infection of Salmonella while the precise mechanisms remain elusive. Autophagy and pyroptosis dependent on the activation of inflammasomes, as parts of innate immune response, contribute to host defense against Salmonella infection. Recently, we reported that spvC could inhibit pyroptosis. To explore the effect of spvC on autophagy and the relationship between its function in pyroptosis and autophagy, infection models of macrophages J774A.1 and epithelial HeLa cells co-cultured with Salmonella Typhimurium wild type, spvC deletion, site-directed mutant which lacks phosphothreonine lyase activity, or complemented strain were established. The levels of LC3 turnover and Beclin 1 of J774A.1 cells were determined by western blot. Confocal laser scanning microscopy was used to visualize the autophagic flux after being transfected with mRFP-GFP-LC3 plasmid in HeLa cells. Results showed that SpvC inhibited autophagosome formation through its phosphothreonine lyase activity. Additionally, analysis of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) and NLR with CARD domain-containing 4 (NLRC4) in J774A.1 cells indicated that spvC decreased the protein levels of NLRP3 and NLRC4, which were significantly changed by autophagy inhibitor Bafilomycin A1. Together, our observations reveal a novel mechanism of spvC in Salmonella pathogenesis and host inflammatory response via inhibiting autophagy and NLRP3 as well as NLRC4. These pathways and their subversion by diverse pathogen virulence determinants are expected to throw light on the design of anti-infective agents.

scholarly journals Palladium nanoparticles induce autophagy and autophagic flux blockade in Hela cells

RSC Advances ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 4130-4141 ◽  
Author(s):  
Li Zhang ◽  
Xuerui Chen ◽  
Jianzhou Wu ◽  
Shiping Ding ◽  
Xu Wang ◽  
...  
Keyword(s):  
Hela Cells ◽  
Palladium Nanoparticles ◽  
Autophagic Flux ◽  
Size Dependent

Size-dependent autophagy and autophagic flux blockade in Hela cells by palladium nanoparticles.

scholarly journals Mutation of the Conserved Threonine 8 within the Human ARF Tumour Suppressor Protein Regulates Autophagy

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 126
Author(s):  
Rosa Fontana ◽  
Daniela Guidone ◽  
Tiziana Angrisano ◽  
Viola Calabrò ◽  
Alessandra Pollice ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Progression ◽  
Hela Cells ◽  
Growth Conditions ◽  
Dual Role ◽  
Tumour Suppressor ◽  
Cellular Stress Response ◽  
Autophagic Flux ◽  
Traditional Role

Background: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. Methods: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. Results: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells’ inability to elicit autophagosomes formation upon T8D expression. Conclusions: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls protein’s ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

scholarly journals ALS/FTD mutations in UBQLN2 impede autophagy by reducing autophagosome acidification through loss of function

2020 ◽  
Vol 117 (26) ◽  
pp. 15230-15241 ◽  
Author(s):  
Josephine J. Wu ◽  
Ashley Cai ◽  
Jessie E. Greenslade ◽  
Nicole R. Higgins ◽  
Cong Fan ◽  
...  
Keyword(s):  
Hela Cells ◽  
Dominant Negative ◽  
Autophagic Flux ◽  
Loss Of Function ◽  
Mutant Proteins ◽  
Function Mechanism ◽  
Important Regulator ◽  
In Vitro Interaction ◽  
Lateral Sclerosis

Mutations inUBQLN2cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALSUBQLN2patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treatUBQLN2-linked ALS/FTD.

Pulsatilla Saponin D Inhibits Autophagic Flux and Synergistically Enhances the Anticancer Activity of Chemotherapeutic Agents Against HeLa Cells

2015 ◽  
Vol 43 (08) ◽  
pp. 1657-1670 ◽  
Author(s):  
Yulin Zhang ◽  
Jiaolin Bao ◽  
Kai Wang ◽  
Xuejing Jia ◽  
Chao Zhang ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Hela Cells ◽  
Anticancer Agent ◽  
Chemotherapeutic Agents ◽  
Autophagic Flux ◽  
Anticancer Activities ◽  
Autophagosome Formation ◽  
Protein Levels ◽  
Pulsatilla Chinensis ◽  
Cancer Types

Pulsatilla saponin D (SB365), a saponin isolated from rhizoma of Pulsatilla chinensis (Bunge) Regel, exhibited anticancer activities in various cancer types. In the present study, we identified that SB365 was a potent inhibitor of autophagic flux in several cancer cell lines. SB365 induced a robust accumulation of autophagosomes as evidenced by monodansylaervarine (MDC) staining and increased protein levels of LC3-II. However, SB365 caused the accumulation of p62, a substrate that should be degraded through the autophagy–lysosomal pathway. These results indicated that SB365 was an inducer of autophagosome formation, but an inhibitor of autophagic flux. Interestingly, we found that SB365 synergistically enhanced the anticancer activity of chemotherapeutic agents against cervical cancer HeLa cells. Furthermore, our study demonstrated that SB365 increased the phosphorylation of ERK and inhibited the phosphorylation of mTOR and p70S6K, suggesting that their roles in the effects of SB365 on autophagy. These results suggest that SB365 could be a promising adjuvant anticancer agent.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

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