scholarly journals Periodic Mechanical Stress Stimulates GIT1-Dependent Mitogenic Signals in Rat Chondrocytes Through ERK1/2 Activity

2018 ◽  
Vol 50 (3) ◽  
pp. 1015-1028 ◽  
Author(s):  
Kewei Ren ◽  
Jilei Tang ◽  
Xuefeng Jiang ◽  
Huiqing Sun ◽  
Luming Nong ◽  
...  
Keyword(s):  
Western Blot ◽  
Mechanical Stress ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Mechanical Stimuli ◽  
Wild Type ◽  
Integrin Β1 ◽  
Chondrocyte Proliferation ◽  
Blocking Antibody

Background/Aims: The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively, but the mechanisms whereby chondrocytes sense and respond to mechanical stimuli remain to be determined. We explored the question and verified the key role of G protein coupled receptor kinase interacting protein 1 (GIT1) signaling in periodic mechanical stress-induced chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to GIT1 or Src or control scrambled shRNA, or transfected with GIT1 wild-type or GIT1 mutant Y321F, or focal adhesion kinase (FAK) wild-type or FAK mutants Y397F or Y576F/Y577, respectively. Moreover, the cells were pretreated with blocking antibody against integrin β1 or PP2. Then the cells were maintained under non-pressure conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of GIT1 at Tyr321. Reduction of GIT1 with shRNA targeted to GIT1 and GIT1 mutant Y321F inhibited periodic mechanical stress-promoted chondrocyte proliferation, accompanied by attenuated extracellular signal-regulated kinase (ERK)1/2 and FAK phosphorylation at Tyr576/577. However, activation of Src and FAK-Tyr397 was not prevented upon GIT1 suppression. Furthermore, pretreatment with blocking antibody against integrin β1, Src-selective inhibitor, PP2, and shRNA targeted to Src blocked GIT1 activation under periodic mechanical stress. In addition, GIT1 phosphorylation at Tyr321 was not reduced upon pretreatment with FAK mutants Y397F or Y576F/Y577 under conditions of periodic mechanical stress. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through at least two separate pathways, integrin β1-Src-GIT1-FAK(Tyr576/577)-ERK1/2, and the other parallel GIT1-independent integrin β1-FAK(Tyr397)-ERK1/2.

scholarly journals Periodic Mechanical Stress Stimulates Cav-1-Dependent IGF-1R Mitogenic Signals in Rat Chondrocytes Through ERK1/2

2018 ◽  
Vol 48 (4) ◽  
pp. 1652-1663 ◽  
Author(s):  
Kewei Ren ◽  
Jilei Tang ◽  
Xuefeng Jiang ◽  
Huiqing Sun ◽  
Luming Nong ◽  
...  
Keyword(s):  
Western Blot ◽  
Mechanical Stress ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biological Effects ◽  
Phosphorylation Site ◽  
Cell Counting ◽  
Mechanical Stimuli ◽  
Chondrocyte Proliferation ◽  
Static Conditions

Background/Aims: The biological effects of periodic mechanical stress on the mitogenesis of chondrocytes have been studied extensively over the past few years. However, the mechanisms underlying the ability of chondrocytes to sense and respond to mechanical stimuli remain to be determined. In the current study, we analyzed the mechanisms by which periodic mechanical stress is translated into biochemical signals and verified the key role of non-integrin mechanosensors including Caveolin-1 (Cav-1), and insulin-like growth factor-1 receptor (IGF-1R) in chondrocyte proliferation. Methods: Two steps were undertaken in the experiment. In the first step, the cells were maintained under static conditions or periodic mechanical stress for 0 h and 1 h prior to Western blot analysis. In the second step, the cells were pretreated with short hairpin RNA (shRNA) targeted to Cav-1 or IGF-1R or control scrambled shRNA. Moreover, they were pretreated with their selective inhibitors methyl β-cyclodextrin (MCD) or Linsitinib (OSI-906). They were maintained under static conditions or periodic mechanical stress for 1 h prior to Western blot analysis, and for 3 days, 8 h per day, prior to direct cell counting and CCK-8 assay, respectively. Results: Periodic mechanical stress significantly induced sustained phosphorylation of Cav-1 at Tyr14 and IGF-1R at Tyr1135/1136. Proliferation was inhibited by pretreatment with Cav-1 inhibitor MCD and by shRNA targeted to Cav-1 in chondrocytes in response to periodic mechanical stress. Meantime, MCD and shRNA targeted to Cav-1 also attenuated IGF-1R, and extracellular signal-regulated kinase (ERK)1/2 activation. In addition, inhibiting IGF-1R activity by Linsitinib and shRNA targeted to IGF-1R abrogated chondrocyte proliferation and phosphorylation level of ERK1/2 subjected to periodic mechanical stress, while the phosphorylation site of Cav-1 was not affected. Conclusion: These findings collectively suggested that periodic mechanical stress promoted chondrocyte proliferation through Cav-1-IGF-1R-ERK1/2.

Functional overlap of the dictyostelium RasG, RasD and RasB proteins

2000 ◽  
Vol 113 (8) ◽  
pp. 1427-1434 ◽  
Author(s):  
M. Khosla ◽  
G.B. Spiegelman ◽  
R. Insall ◽  
G. Weeks
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ras Proteins ◽  
Wild Type ◽  
Vegetative Cells ◽  
Protein Levels ◽  
Ras Genes ◽  
Downstream Effector ◽  
In The Wild

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(-)cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(-)cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(-)cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(-)cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

Carboplatin-induced Senescence in Ovarian Cancer Cells Is Reversed Through EGFR and NF-κB Signaling

2021 ◽  
Author(s):  
Yue Li ◽  
Mingxu Fu ◽  
Ling Guo ◽  
Xiaoxiao Sun ◽  
Yuhang Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Blue Staining ◽  
Ovarian Cancer Cells ◽  
Positive Staining ◽  
The Impact

Abstract Background: Metastases and recurrence of ovarian cancer after surgery and chemotherapy account for most cancer-related deaths, yet the mechanism underlying metastases and recurrence remains poorly understood. Recent evidence demonstrates that although long-lasting cells were considered tumor suppressors, senescent cancer cells, can induce the metastases and recurrence. In this study, we focused on the fate of ovarian cancer cells treated with carboplatin and explored the mechanism underlying ovarian cancer cell recovery from chemotherapy-induced senescence. Methods: SÁ-β-galactosidase staining was used to detect the impact of carboplatin on senescence of ovarian cancer cells. Cell proliferation was determined using direct cell counting, clone formation assay and 3D tumor spheroid formation assay. Lentivirus-mediated transduction was used to silence or upregulate EGFR expression. Quantitative real-time PCR and western blot analysis validated the efficacy of the knockdown or overexpression effect. Immunofluorescence staining and western blot analysis were used to examined the expression of EGFR and NF-KB. Cell death was determined using trypan blue staining assay. Results: Ovarian cancer cells treated by carboplatin exhibit a senescence-like phenotype indicated by SA-β-galactosidase positive staining. Importantly, carboplatin-induced senescence-like phenotype is reversible. In ovarian cancer cells, EGFR positively regulated cells proliferation, decreased carboplatin-induced senescence and upregulated the NF-κB1 protein level. EGFR/NF-κB1 upregulation promoted the recovery of ovarian cancer cells from senescence and chemoresistance to carboplatin. Conclusions: Ovarian cancer cells treated with carboplatin displayed a reversible senescence-like phenotype that could be combined with EGFR or NF-κB1 inhibitors to improve treatment effects.

The CALM/AF10 Interactor CATS Is a Substrate of KIS, a Positive Regulator of Cell Cycle Progression in Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2549-2549
Author(s):  
Leticia Fröhlich Archangelo ◽  
Fabíola Traina ◽  
Philipp A Greif ◽  
Alexandre Maucuer ◽  
Valérie Manceau ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Cycle Progression

Abstract Abstract 2549 The CATS protein (also known as FAM64A and RCS1) was first identified as a novel CALM (PICALM) interactor that interacts with and influences the subcellular localization of CALM/AF10, a leukemic fusion protein found in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and in malignant lymphoma. CATS is highly expressed in leukemia, lymphoma and tumor cell lines but not in non-proliferating T-cells or in peripheral blood lymphocytes (PBLs). The protein levels of CATS are cell cycle-dependent, induced by mitogens (e.g. PHA) and correlate with the proliferative state of the cell. Thus, CATS is as a marker for proliferation. Using CATS as a bait in a yeast two-hybrid screen we identified the Kinase Interacting Stathmin (KIS or UHMK1) as a CATS interacting partner. KIS is a serine/threonine kinase that positively regulates cell cycle progression through phosphorylation of p27KIP in leukemia cell lines. The interaction between CATS and KIS was confirmed by GST pull-down, and co-immunopreciptation. KIS interaction region was mapped to CATS N-terminal portion. Searching through the phosphorylation site databases PhosphoSitePlus™ (http://www.phosphosite.org) and Phosida (http://www.phosida.com/) we identified 9 residues within CATS shown to be subject of post-translational modification. Phosphorylation assay with recombinant KIS demonstrated that this kinase efficiently phosphorylated full length CATS and its N-terminal part, but not the C-terminal of the protein. To map the KIS phosphorylation site of CATS, peptides comprising all known phospho-sites of CATS N-terminal (S16, S129, S131, T133 and S135) and mutations of the putative KIS target motif (S129 and S131) were tested for KIS phosphorylation. Thereby, we identified CATS S131 as the unique target site for KIS phosphorylation. Western blot analysis of U2OS cells, which had undergone cell cycle synchronization by a double thymidine block, revealed that KIS fluctuated throughout the cell cycle and counteracted CATS levels. Furthermore, we analyzed KIS protein expression on bone marrow mononuclear cells (MNCs) of MDS and AML patients. We studied 5 healthy donors, 13 MDS patients (7 low-risk [RA/RARS] and 6 high-risk [RAEB/RAEBt] according to FAB classification) and 10 AML patients (7 de novo and 3 secondary). Western blot analysis revealed elevated levels of KIS in MDS and AML compared to the control samples. We used a reporter gene assay in order to determine the influence of KIS on the CATS-mediated transcriptional repression and to elucidate the role of KIS-dependent phosphorylation of CATS at serine 131 in this context. Coexpression of GAL4-DBD-CATS and KIS enhanced the inhibitory function of CATS on transactivation of the GAL4-tk-luciferase reporter. This effect of KIS was observed for both CATS wild type and CATS phospho-defective mutant (CATS S131A) but not when the kinase dead mutant KISK54R was used. Moreover, CATS phosphomimetic clone (CATSS131D) exerted the same transcriptional activity as the CATS wild type. These results demonstrate that KIS enhances the transcriptional repressor activity of CATS, and this effect is independent of CATS phosphorylation at S131 but dependent on the kinase activity of KIS. Finally, we investigated whether CATS would affect the CALM/AF10 function as an aberrant transcription factor. Coexpression of constant amounts of GAL4-DBD-CALM/AF10 and increasing amounts of CATS lead to reduced transactivation capacity of CALM/AF10 in a dose dependent manner. Our results show that CATS not only interacts with but is also a substrate for KIS, suggesting that CATS function might be modulated through phosphorylation events. The identification of the CATS-KIS interaction further supports the hypothesis that CATS plays an important role in the control of cell proliferation. Moreover the elevated levels of KIS in hematological malignances suggest that KIS could regulate CATS activity and/or function in highly proliferating leukemic cells. Thus our results indicate that CATS function might be important to understand the malignant transformation mediated by CALM/AF10. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The role of activating transcription factor 6 in hydroxycamptothecin-induced fibroblast autophagy and apoptosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jin Tao ◽  
Hui Chen ◽  
Xiaolei Li ◽  
Jingcheng Wang
Keyword(s):  
Transcription Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Immunofluorescent Staining ◽  
Fibroblast Proliferation ◽  
Activating Transcription Factor ◽  
Related Proteins

Abstract Background The over-proliferation of fibroblasts is considered to be the main cause of scar adhesion after joint surgery. Hydroxycamptothecin (HCPT), though as a potent antineoplastic drug, shows preventive effects on scar adhesion. This study aimed to investigate the role of activating transcription factor 6 (ATF-6) in the HCPT-induced inhibition of fibroblast viability. Methods The cell counting kit-8 (CCK-8) assay, western blot analysis, lentivirus-mediated gene silencing, transmission electron microscopy (TEM) analysis, immunofluorescent staining for autophagy-related protein light chain 3 (LC3) were used to explore the effect of HCPT on triggering fibroblast apoptosis and inhibiting fibroblast proliferation, and the involvement of possible signaling pathways. Results It was found that HCPT exacerbated fibroblast apoptosis and repressed its proliferation. Subsequently, endoplasmic reticulum stress (ERS)-related proteins were determined by western blot prior to ATF6 p50 was screened out and reexamined after it was silenced. As a result, ATF6-mediated ERS played a role in HCPT-induced fibroblast apoptosis. Autophagy-related proteins and autophagosomes were detected after the HCPT administration using western blot and TEM analyses, respectively. Autophagy was activated after the HCPT treatment. With the co-treatment of autophagy inhibitor 3-methyladenine (3-MA), both the western blot analysis and the CCK-8 assay showed inhibited autophagy, which indicated that the effect of HCPT on fibroblast proliferation was partially reversed. Besides, the LC3 immunofluorescence staining revealed suppressed autophagy after silencing ATF6 p50. Conclusion Our results demonstrate that HCPT acts as a facilitator of fibroblast apoptosis and inhibitor of fibroblast proliferation for curbing the postoperative scar adhesion, in which the ATF6-mediated ERS pathway and autophagy are involved.

scholarly journals Modulation of Long-Term Potentiation by Gamma Frequency Transcranial Alternating Current Stimulation in Transgenic Mouse Models of Alzheimer’s Disease

2021 ◽  
Vol 11 (11) ◽  
pp. 1532
Author(s):  
Won-Hyeong Jeong ◽  
Wang-In Kim ◽  
Jin-Won Lee ◽  
Hyeng-Kyu Park ◽  
Min-Keun Song ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Western Blot ◽  
Mouse Models ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Alternating Current ◽  
Wild Type ◽  
Gamma Frequency ◽  
Transcranial Alternating Current Stimulation ◽  
5Xfad Mice

Transcranial alternating current stimulation (tACS) is a neuromodulation procedure that is currently studied for the purpose of improving cognitive function in various diseases. A few studies have shown positive effects of tACS in Alzheimer’s disease (AD). However, the mechanism underlying tACS has not been established. The purpose of this study was to investigate the mechanism of tACS in five familial AD mutation (5xFAD) mouse models. We prepared twenty 4-month-old mice and divided them into four groups: wild-type mice without stimulation (WT-NT group), wild-type mice with tACS (WT-T group), 5xFAD mice without stimulation (AD-NT group), and 5xFAD mice with tACS (AD-T group). The protocol implemented was as follows: gamma frequency 200 μA over the bilateral frontal lobe for 20 min over 2 weeks. The following tests were conducted: excitatory postsynaptic potential (EPSP) recording, Western blot analysis (cyclic AMP response element-binding (CREB) proteins, phosphorylated CREB proteins, brain-derived neurotrophic factor, and parvalbumin) to examine the synaptic plasticity. The EPSP was remarkably increased in the AD-T group compared with in the AD-NT group. In the Western blot analysis, the differences among the groups were not significant. Hence, tACS can affect the long-lasting enhancement of synaptic transmission in mice models of AD.

scholarly journals The mechanisms of fructose 1,6 bisphosphatase degradation in methylotrophic yeasts Pichia pastoris

2018 ◽  
Vol 22 ◽  
pp. 235-239
Author(s):  
O. V. Dmytruk ◽  
N. V. Bulbotka ◽  
A. A. Sibirny
Keyword(s):  
Pichia Pastoris ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Activity ◽  
Wild Type ◽  
Methylotrophic Yeasts ◽  
Methanol Induction ◽  
Fructose 1,6 Bisphosphatase ◽  
In The Wild

Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme. Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.

A Novel DNM2 Mutation Displaying Embryonic Lethality and Impaired Transferrin Uptake Identified in a Mouse ENU Mutagenesis Screen for Genes Perturbing Erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 608-608 ◽  
Author(s):  
Fiona C Brown ◽  
Michael Collett ◽  
Phillip J Robinson ◽  
James C Whisstock ◽  
Douglas J. Hilton ◽  
...  
Keyword(s):  
Iron Deficiency ◽  
Western Blot ◽  
Blot Analysis ◽  
Human Disease ◽  
Western Blot Analysis ◽  
Deficiency Anemia ◽  
Wild Type ◽  
Enu Mutagenesis ◽  
Mutagenesis Screen ◽  
Transferrin Uptake

Abstract Abstract 608 Forward-genetic screens have become a powerful method to study the pathogenesis of human disease and gene function. Chemical mutagenesis in mice using the mutagen, N-ethyl N-nitrosourea (ENU), has shown to be highly successful in elucidating novel genes or alleles in a variety of biological pathways, describing new functions of existing genes, and establishing mouse models that accurately recapitulate human disease. Advances in mapping strategies and deep sequencing technologies has dramatically simplified mutation detection, making ENU mutagenesis screens a feasible tool to study specific organ systems. To identify novel alleles regulating erythropoiesis, our laboratory has undertaken a dominant ENU mutagenesis screen. In this screen, the G1 progeny were screened at seven weeks of age for abnormalities in red cell indices (MCV, MCH, and HCT) using an automated hematological analyser. Here, we describe the identification of mice with a missense mutation of the large GTPase Dynamin 2 (DNM2) leading to an amino acid substitution V235G, predicted to lie within the nucleotide binding pocket for GTP. Western blot analysis for DNM2 protein revealed 50% protein levels in heterozygotes, suggesting that the point mutation leads to loss of protein rather than a dominant negative effect. Inherited DNM2 mutations are associated with autosomal dominant Charcot Tooth Myopathy (CTM) and Centronuclear Myopathy (CNM), but no recognised blood disorders. Heterozygous DNM2V235G displayed hypochromic, microcytic anemia – HGB (15 g/dl compared to 16.5 g/dl in wild type mice), MCV (41.3 fl compared to 45.6 fl in wild type mice) and MCH (12.7 pg compared to 14.5 pg in wild type mice), but no obvious neuropathy or myopathy. Homozygosity was lethal before embryonic (E) day 8.5. DNM2 is an essential component in clathrin-mediated endocytosis, which is required for uptake of transferrin into red cells for incorporation of heme. Accordingly, endocytosis assays for transferrin uptake by FACS and confocal microscopy revealed reduced uptake in heterozygotes, explaining the microcytic hypochromic anemia. Western blot analysis for ferritin demonstrated reduced cellular ferritin, indicating cellular iron deficiency. Thus, this mouse model provides the first in vivo evidence that haplo-insufficiency of DNM2 can lead to iron deficiency anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Critical Roles of the PI3K-Akt-mTOR Signaling Pathway in Apoptosis and Autophagy of Astrocytes Induced by Methamphetamine

2019 ◽  
Vol 17 (1) ◽  
pp. 96-104 ◽  
Author(s):  
Han-Qing Liu ◽  
Ya-Wen An ◽  
A-Zhen Hu ◽  
Ming-Hua Li ◽  
Jue-Lian Wu ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Signaling ◽  
Cell Counting ◽  
Mtor Signaling Pathway ◽  
Induced Apoptosis

AbstractThis study aimed to reveal potential roles of the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway in apoptosis and autophagy of astrocytes induced by methamphetamine (METH). A Cell Counting Kit-8 (CCK-8) was used to determine the reduction in proliferation of U-118 MG cells induced by METH. Hoechst 33258 and flow cytometry were used to observe the astrocytes. Western blot analysis was performed to evaluate protein expression and phosphorylation levels. METH inhibited the proliferation of U-118 MG cells and induced apoptosis and autophagy. Western blot analysis showed that the ratio of LC3-II/I was increased, whereas the expression of Bcl-2 was decreased. The phosphorylation cascade of kinases in the PI3K-Akt-mTOR signaling pathway was significantly inhibited by METH exposure, as were proteins downstream of mTORC1, such as p70s6k, rps6, 4EBP1 and eIF4E. METH inhibited proliferation of U-118 MG cells and induced apoptosis and autophagy. The PI3K-Akt-mTOR signaling pathway likely plays a critical role in these effects.

scholarly journals Paeonol Induces Protective Autophagy in Retinal Photoreceptor Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Daowei Zhang ◽  
Jiawen Wu ◽  
Jihong Wu ◽  
Shenghai Zhang
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Retinal Photoreceptor

Background: Retinal photoreceptor (RP) cells are widely involved in retina-related diseases, and oxidative stress plays a critical role in retinal secondary damage. Herein, we investigated the effectiveness and potential mechanisms of autophagy of paeonol (Pae) in terms of oxidation resistance.Methods: The animal model was induced by light damage (LD) in vivo, whereas the in vitro model was established by H2O2 stimulation. The effectiveness of Pae was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence, transmission electron microscopy, electroretinogram, and Western blot analysis in vivo, and the underlying mechanisms of Pae were assessed by Cell Counting Kit-8 assay, reactive oxygen species (ROS) assay, and Western blot analysis in 661W cells. We mainly evaluated the effects of Pae on apoptosis and autophagy.Results: Increased apoptosis of the LD-induced and decreased autophagy of RPs were mitigated by Pae treatment. Pea, which increased the expression of mitochondrial functional protein cytochrome c, reversed the decreased cell viability and autophagy induced by oxidative stress in 661W cells. Experiments showed that autophagy was downregulated in PINK1/Parkin dependent and the BNIP3L/Nix dependent pathways under H2O2 stimulation and was upregulated by Pae treatment. Pae increased the cell viability and reduced ROS levels through autophagy.Conclusion: Pretreatment with Pae preserved RP cells by enhancing autophagy, which protected retinal function.

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