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.

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.

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 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 Dysregulation of CLOCK gene expression in hyperoxia-induced lung injury

2014 ◽  
Vol 306 (11) ◽  
pp. C999-C1007 ◽  
Author(s):  
Venu Lagishetty ◽  
Prasanna Tamarapu Parthasarathy ◽  
Oluwakemi Phillips ◽  
Jutaro Fukumoto ◽  
Young Cho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lung Injury ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Clock Gene ◽  
Target Genes ◽  
Clock Genes ◽  
Wild Type ◽  
Clock Gene Expression

Hyperoxic acute lung injury (HALI) is characterized by inflammation and epithelial cell death. CLOCK genes are master regulators of circadian rhythm also implicated in inflammation and lung diseases. However, the relationship of CLOCK genes in hyperoxia-induced lung injury has not been studied. This study will determine if HALI alters CLOCK gene expression. To test this, wild-type and NALP3−/− mice were exposed to room air or hyperoxia for 24, 48, or 72 h. In addition, mice were exposed to different concentrations of hyperoxia (50, 75, or 100% O2) or room air for 72 h. The mRNA and protein levels of lung CLOCK genes, based on quantitative PCR and Western blot analysis, respectively, and their target genes are significantly elevated in mice exposed to hyperoxia compared with controls. Alterations in CLOCK genes are associated with increased inflammatory markers in bronchoalveolar lavage fluid of hyperoxic mice compared with controls. Histological examination of mice lungs exposed to hyperoxia show increased inflammation and alveolar congestion compared with controls. Our results indicate sequential increase in CLOCK gene expression in lungs of mice exposed to hyperoxia compared with controls. Additionally, data suggest a dose-dependent increase in CLOCK gene expression with increased oxygen concentrations. To validate if the expression changes related to CLOCK genes are indeed associated with inflammation, NALP3−/− was introduced to analyze loss of function in inflammation. Western blot analysis showed significant CLOCK gene downregulation in NALP3−/− mice compared with wild-type controls. Together, our results demonstrate that hyperoxia-mediated lung inflammation is associated with alterations in CLOCK gene expression.

scholarly journals Identification and Characterization of Epp, the Secreted Processing Protease for the Vibrio anguillarum EmpA Metalloprotease

2008 ◽  
Vol 190 (20) ◽  
pp. 6589-6597 ◽  
Author(s):  
Maureen Varina ◽  
Steven M. Denkin ◽  
Andrew M. Staroscik ◽  
David R. Nelson
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Protease Activity ◽  
Western Blot Analysis ◽  
Culture Supernatant ◽  
Vibrio Anguillarum ◽  
Wild Type ◽  
Processing Protease ◽  
Culture Supernatants

ABSTRACT The zinc metalloprotease EmpA is a virulence factor for the fish pathogen Vibrio anguillarum. Previous studies demonstrated that EmpA is secreted as a 46-kDa proenzyme that is activated extracellularly by the removal of an ∼10-kDa propeptide. We hypothesized that a specific protease is responsible for processing secreted pro-EmpA into mature EmpA. To identify the protease responsible for processing pro-EmpA, a minitransposon mutagenesis (using mini-Tn10Km) clone bank of V. anguillarum was screened for reduced protease activity due to insertions in undescribed genes. One mutant with reduced protease activity was identified. The region containing the mini-Tn10Km was cloned, sequenced, and found to contain epp, an open reading frame encoding a putative protease. Further characterization of epp was done using strain M101, created by single-crossover insertional mutagenesis. Protease activity was absent in M101 cultures even when empA protease activity was induced by salmon gastrointestinal mucus. When the epp mutation was complemented with a wild-type copy of epp (M102), protease activity was restored. Western blot analysis of sterile filtered culture supernatants from wild-type (M93Sm) cells, M101 cells, and M102 cells revealed that only pro-EmpA was present in M101supernatants; both pro-EmpA and mature EmpA were detected in M93Sm and M102 supernatants. When sterile filtered culture supernatants from the empA mutant strain (M99) and M101 were mixed, protease activity was restored. Western blot analysis revealed that pro-EmpA in M101 culture supernatant was processed to mature EmpA only after mixing with M99 culture supernatant. These data show that Epp is the EmpA-processing protease.

scholarly journals Delta-Aminolevulinate Preferentially Induces Gamma-Globin Expression in Erythroid Cells through Activation of NRF2 Stress Response

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3387-3387
Author(s):  
Li Liu ◽  
Christina Torres ◽  
Baolin Kang ◽  
Betty Pace
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Heme Biosynthesis ◽  
Luciferase Reporter ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Ku812 Cells ◽  
Globin Promoter

Abstract Fetal hemoglobin (HbF,α2γ2) is an important genetic modifier for β-hemoglobinopathies including sickle cell disease (SCD) and β-Thalassemia which occur as a result of mutations in the adult β-globin gene. δ-aminolevulinate (ALA), the first product synthesized in the mammalian heme biosynthesis pathway, has been reported to induce hemoglobin production. In this study, we determined the effects of ALA on γ- and β-globin transcription and HbF production in an erythroid cell line and primary erythroid progenitors. We demonstrated that ALA increased heme levels by 50% with 48 hour treatment in KU812 erythroleukemia cells. By reverse transcription-real time polymerase chain reaction (RT-qPCR), we detected an induction of γ-globin transcription in a time- and dose-dependent manner with 2.4-fold elevation at the 2mM ALA concentration. Moreover, we showed that HbF protein level increased dramatically by 22.9-fold at 48 hour ALA treatment by western blot analysis with HbF specific antibody. By contrast, ALA treatment did not change β-globin transcription level. Subsequent studies in primary erythroid progenitors derived from CD34+ stem cells, we observed γ-globin transcriptional activation up to 6.5-fold by day 22 in culture with 48hour ALA treatment, whereas β-globin transcription was not significantly changed. Studies to determine molecular mechanisms demonstrated that addition of succinylacetone (SA), a specific inhibitor of heme biosynthesis, blocked the ALA-mediated induction of γ-globin transcription and HbF synthesis in KU812 cells detected by RT-qPCR, western blot and enzyme-linked immunosorbent assay. Using 2'-7'-Dichlorodihydrofluorescein diacetate with flow cytometer detection, we observed a 3-fold increase of reactive oxygen species such as hydrogen peroxide with 24-hour ALA treatment in KU812 cells which was inhibited by the addition of SA. In addition, western blot analysis detected increased nuclear translocation of NRF2, a master transcription factor controlling the cellular antioxidant response. Using chromatin immunoprecipitation, we demonstrated that NRF2-associated enrichment of γ-globin proximal promoter chromatin was enhanced 10-fold in the region of the antioxidant responsive element. Furthermore, in KU812 cells transiently transfected with a wild-type and ARE-mutated γ-globin promoter luciferase reporters followed by ALA treatment, we detected activation of the wild-type γ-globin promoter-driven luciferase reporter. As predicted, deletion of the ARE in the γ-globin promoter not only significantly reduced the basal activity by 10-fold but also abolished the ALA-induced promoter activity. These data support a role of ALA in activating heme production and γ-globin expression through an NRF2 activation mechanism. Our results provide evidence that ALA and the heme biosynthesis pathway are novel targets for therapeutic potential in treating β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

scholarly journals A 36 residues insertion in the dimerization domain of the growth hormone receptor results in defective trafficking rather than impaired signaling

2006 ◽  
Vol 188 (2) ◽  
pp. 251-261 ◽  
Author(s):  
M Maamra ◽  
A Milward ◽  
H Zarkesh Esfahani ◽  
L P Abbott ◽  
L A Metherell ◽  
...  
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Cell Surface ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mammalian Cells ◽  
Growth Hormone Receptor ◽  
Wild Type ◽  
Dimerization Domain

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.

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.

Rab7b suppresses autophagy and induces apoptosis of K562 leukemic cells treated with homoharringtonine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4884-4884 ◽  
Author(s):  
Peilin Lu ◽  
Donghua He ◽  
Yang Yang ◽  
Pengfei Hu ◽  
Yi Zhao ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Rab Proteins ◽  
Wild Type ◽  
Akt Phosphorylation

Abstract Abstract 4884 Homoharringtonine is an effective anti-leukemia medicine developed by Chinese. It has been found to induce differentiation and apoptosis of leukemia cells. However, the mechanism of its anti-leukemia function has not been fully understood. Rab is a kind of G protein of Ras superfamily and participates in endocytosis and exocytosis of protein transport process. In recent studies, some Rab proteins such as Rab7 are found to be associated with cellular autophagy and apoptosis. We previously identified a new small GTPase homologous to Rab7, named Rab7b, which is selectively expressed in promyeloid and monocytic cells and is localized to lysosome-associated compartments. To investigate the roles of Rab7b in acute myeloid leukemia, we used leukemia cell line K562 as a model in the present study. After treatment of K562 cells with various doses of HHT, cell viability and apoptosis were measured by MTT assay and Annexin V/PI staining respectively. Protein expression of LC3, a marker of autophagy, and caspase3, 9, ERK1/2,Akt were determined by Western blot analysis. Using stable gene transfection, several Rab7b variants, including Rab7b wild-type, active mutant Rab7b-Q67L and localization-deficient mutant Rab7b-ΔCC as well as Rab7b RNAi were transfected in to K562 cells and their roles in regulation of apoptosis in K562 leukemia cells induced by HHT were further evaluated. Our data showed that the viability of the K562 cells was greatly reduced by HHT treatment in a dose- and time- dependant manner. Treatment of the K562 with HHT significantly increases apoptosis in the cells as measured by Annexin V/PI staining. Using Western blot analysis, we further determined that the expression of caspase3, 9 was increased, and ERK1/2 augmented with Akt was suppressed in the cells treated with HHT. After suppressing autophagy with 3-MA, apoptosis was enhanced in the K562 cells treated with HHT. (p<0.05). By constitutively expression of Rab7B and variants in K562 cells, we found that the rate of apoptotic cells are much higher in the K562 cells transfected with Rab7b wild-type and Rab7b-Q67L variants, along with increased expression of caspase3, 9, ERK1/2 and decreased expression of Akt in the transfectants with Rab7b wild-type and active mutant Rab7b-Q67L. Our study suggests that HHT is able to suppress autophagy and enhance apoptosis in K562 leukemia cells in a caspase-dependent way, which is associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Over-expression of Rab7b can enhance HHT induced apoptosis in K562 cells, which may also be associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Taken together, our study elucidates a new recognition for the mechanism of HHT in anti-leukemia therapy and provides a new insight into understanding the relationship between autophagy and apoptosis in leukemia cells induced chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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