scholarly journals Proteasome (Prosome) Subunit Variations during the Differentiation of Myeloid U937 Cells

1997 ◽  
Vol 15 (3) ◽  
pp. 131-144 ◽  
Author(s):  
Laurent Henry ◽  
Ahsene Baz ◽  
Marie-Thérèse Château ◽  
René Caravano ◽  
Klaus Scherrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Western Blot ◽  
Control Level ◽  
U937 Cells ◽  
Myristate Acetate ◽  
Multicatalytic Proteinase ◽  
Undifferentiated Cells ◽  
Proteasome Subunits ◽  
Protein Particles

20S proteasomes (prosomes/multicatalytic proteinase) are protein particles built of 28 subunits in variable composition. We studied the changes in proteasome subunit composition during the differentiation of U937 cells induced by phorbol‐myristate‐acetate or retinoic acid plus 1,25‐dihydroxy‐cholecalciferol by western blot, flow cytometry and immuno‐fluorescence. p25K (C3), p27K (IOTA) and p30/33K (C2) subunits were detected in both the nucleus and cytoplasm of undifferentiated cells. Flow cytometry demonstrated a biphasic decrease in proteasome subunits detection during differentiation induced by RA+VD. PMA caused an early transient decrease in these subunits followed by a return to their control level, except for p30/33K, which remained low. Immuno‐fluorescence also showed differences in the cytolocalization of the subunits, with a particular decrease in antigen labeling in the nucleus of RA+VD‐induced cells, and a scattering in the cytoplasm and a reorganization in the nucleus of PMA‐induced cells. Small amounts of proteasomal proteins were seen on the outer membrane of non‐induced cells; these membrane proteins disappeared when treated with RA+VD, whereas some increased on PMA‐induced cells. The differential changes in the distribution and type of proteasomes in RA+VD and PMA‐induced cells indicate that, possibly, 20S proteasomes may play a role in relation to the mechanisms of differentiation and the inducer used.

scholarly journals Effects and Mechanism of Oxymatrine Combined with Compound Yinchen Granules on the Apoptosis of Hepatocytes through the Akt/FoxO3a/Bim Pathway

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Author(s):  
Xian Zhang ◽  
Jiajia Ge ◽  
Xuejuan Zhu ◽  
Haifeng Zhang ◽  
Yuanzi Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Liver Failure ◽  
Acute Liver Failure ◽  
Rat Liver ◽  
Caspase 3 ◽  
Normal Group ◽  
Model Group ◽  
Apoptosis Rate ◽  
Liver Tissues

The aim of the present study was to investigate the effects and mechanism of oxymatrine (OMT) combined with compound yinchen granules (CYG) on the apoptosis of hepatocytes through the Akt/FoxO3a/Bim pathway in rats with acute liver failure. The rat model of acute liver failure was established using lipopolysaccharide/D-galactosamine (LPS/D-GalN). The expression of proteins in rat liver tissues was detected by western blot analysis. The mRNA expression of FoxO3a, Bim, Bax, Bcl-2, and caspase-3 in rat liver tissues was detected by RT-qPCR. The apoptosis rate of rat hepatocytes was determined by flow cytometry. Western blots showed that when compared with the normal group, the expression of p-Akt and p-FoxO3a in the model group was decreased ( P < 0.05 ), while the expression of Bim was increased ( P < 0.01 ). Compared with the model group, the expression of p-Akt and p-FoxO3a in the OMT group and the OMT combined with CYG groups was increased ( P < 0.05 or P < 0.01 ), while the expression of Bim was decreased ( P < 0.05 ). The Bax/Bcl-2 ratio and caspase-3 protein expression in the model group were significantly higher than those in the normal group ( P < 0.01 ). The Bax/Bcl-2 ratio and the expression of caspase-3 protein in the OMT group and the OMT combined with CYG groups were significantly lower than those in the model group ( P < 0.01 ). The results of RT-qPCR were consistent with those of western blot. The results of flow cytometry showed that the apoptosis rate of hepatocytes in the OMT group and the OMT combined with CYG groups was significantly lower than that in the model group ( P < 0.05 or P < 0.01 ). We concluded that LPS/D-GalN can induce apoptosis of hepatocytes in rats with acute liver failure through the Akt/FoxO3a/Bim pathway. OMT combined with CYG inhibits apoptosis of hepatocytes in rats with acute liver failure via the Akt/FoxO3a/Bim pathway.

scholarly journals Polyporus polysaccharide regulates proliferation, apoptosis and migration of bladder cancer cells by polarizing macrophages to M1 subtype in tumor microenvironment

2020 ◽  
Author(s):  
Wenyu Jia ◽  
Siwan Luo ◽  
Gena Lai ◽  
Shiqi Li ◽  
Shuai Huo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Bladder Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Activated Macrophages ◽  
Tnf Α ◽  
Bladder Cancer Cells ◽  
And Migration ◽  
Mesenchymal Transformation ◽  
M1 Type

Abstract BackgroundPolyporus polysaccharide (PPS), an active ingredient of traditional Chinese medicinal Polyporus umbellatus, has multiple biological functions, such as anti-cancer, immune-regulating and hepatoprotective activities. The purpose of this study was to investigate the mechanism of PPS activated macrophages in the treatment of bladder cancer.Methods100 ng/mL Phorbol myristate acetate (PMA) was used to induce THP-1 human leukemic cells as a macrophage model. Flow cytometry was used to detect the expression of CD14 and CD68 to verify the establishment of macrophage model. After that, Macrophages derived from THP-1 were treated with different concentrations of PPS (1,10 and 100 ug/mL). Flow cytometry and RT-PCR were used to detected the expression of CD16, CD23, CD86, CD40 and interleukin (IL)-Iβ, iNOS mRNA. ELISA was used to test the change of IL-1β and TNF-α in macrophage after the treatment with PPS. The conditioned medium from PPS-polarized macrophages was used to detect the effect of activated macrophages on bladder cancer. MTT assay, 5-ethynyl-2¢-deoxyuridine assay, flow cytometry, Transwell assay, and Western blot analysis were used to detect the effects of polarized macrophages on the viability, proliferation, apoptosis, and migration of bladder cancer cells. Western blot was also used to analysis the change of JAK2/NF-κB pathway protein.ResultsPPS promoted the expression of pro-inflammatory factors, such as IL-Iβ, TNF-α and iNOS, and surface molecules CD86, CD16, CD23, and CD40 in macrophages and then polarized macrophages to M1 type. The results demonstrated that activated macrophages inhibited the proliferation of bladder cancer cells, regulated their apoptosis, and inhibited migration and epithelial–mesenchymal transformation (EMT). JAK2/NF-κB pathways were downregulated in the anti-bladder cancer process of activated macrophages. ConclusionThe findings indicated that PPS inhibited the proliferation and progression of bladder cancer by the polarization of macrophages to M1 type, and JAK2/NF-κB pathway was downregulated in the process of anti-bladder cancer.

scholarly journals Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246197
Author(s):  
Jorge Marquez ◽  
Jianping Dong ◽  
Chun Dong ◽  
Changsheng Tian ◽  
Ginette Serrero
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Negative Regulator ◽  
Target Validation ◽  
Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

The binding epitope of sintilimab on PD-1 revealed by AbMap

2021 ◽  
Author(s):  
Mingliang Ma ◽  
Huan Qi ◽  
Chuansheng Hu ◽  
Zhaowei Xu ◽  
Fanlin Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Structural Analysis ◽  
Western Blot ◽  
Epitope Mapping ◽  
High Efficiency ◽  
Point Mutations ◽  
Therapeutic Antibodies ◽  
Binding Interface ◽  
Binding Epitope ◽  
Key Residues

Abstract PD-1 plays an important role as an immune checkpoint. Sintilimab is a newly approved PD-1 antibody for cancer immunotherapy with an unknown binding epitope on PD-1. In this study, to elucidate the molecular mechanism by which sintilimab blocks PD-1 activation, we applied Antibody binding epitope Mapping (AbMap) to identify the binding epitope of sintilimab. An epitope was successfully identified, i.e. SLAPKA, aa 127–132. By constructing a series of point mutations, the dominant residues S127, L128, A129, P130, and A132 of PD-1 were further validated by western blot analysis, biolayer interferometry, and flow cytometry. Structural analysis showed that the epitope is partially within the binding interface of PD-1 and PD-L1, and this epitope also partially overlaps with that of nivolumab and pembrolizumab. These results demonstrate that sintilimab can attenuate PD-1 activation by directly competing with the interaction between PD-1 and PD-L1 through binding with the key residues of the FG loop on PD-1. This study also demonstrates the high efficiency and accuracy of AbMap for determining the binding epitope of therapeutic antibodies.

Abstract 241: Retinoic Acid Promotes Metabolic Maturation of Human Embryonic Stem Cell-derived Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Shumei Miao ◽  
Xing Fang ◽  
Xiaoxiao Wang ◽  
Lingqun Ye ◽  
Jingsi Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Heart Development ◽  
Drug Testing ◽  
Disease Modeling ◽  
Time Window ◽  
Embryonic Stem ◽  
Early Time ◽  
Control Group ◽  
Cardiomyocyte Differentiation

Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development, and studies on RA can provide clues for understanding cardiomyocyte differentiation and maturation. In this study, we studied the roles of RA during cardiomyocyte differentiation and maturation, systematically. After adding RA at different stages of cardiomyocyte differentiation, we compared the efficiency of differentiation by quantitative real-time PCR and flow cytometry. We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2, NKX2.5 and MYH6 on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. Furthermore, RA was added at different time intervals after purification to induce cardiomyocyte maturation. Our results demonstrated that RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number, and promoted RNA splicing switch. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine. In conclusion, our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte metabolic maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.

scholarly journals A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

Antibodies ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 37 ◽  
Author(s):  
Jennifer Linden ◽  
Kiel Telesford ◽  
Samantha Shetty ◽  
Paige Winokour ◽  
Sylvia Haigh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Monoclonal Antibodies ◽  
Western Blot ◽  
Clostridium Perfringens ◽  
Specificity And Sensitivity ◽  
Epsilon Toxin ◽  
Blocking Antibodies

The pore-forming epsilon toxin (ETX) produced by Clostridium perfringens is among the most lethal bacterial toxins known. Sensitive antibody-based reagents are needed to detect toxin, distinguish mechanisms of cell death, and prevent ETX toxicity. Using B-cell immuno-panning and cloning techniques, seven ETX-specific monoclonal antibodies were generated from immunized rabbits. ETX specificity and sensitivity were evaluated via western blot, ELISA, immunocytochemistry (ICC), and flow cytometry. ETX-neutralizing function was evaluated both in vitro and in vivo. All antibodies recognized both purified ETX and epsilon protoxin via western blot with two capable of detecting the ETX-oligomer complex. Four antibodies detected ETX via ELISA and three detected ETX bound to cells via ICC or flow cytometry. Several antibodies prevented ETX-induced cell death by either preventing ETX binding or by blocking ETX oligomerization. Antibodies that blocked ETX oligomerization inhibited ETX endocytosis and cellular vacuolation. Importantly, one of the oligomerization-blocking antibodies was able to protect against ETX-induced death post-ETX exposure in vitro and in vivo. Here we describe the production of a panel of rabbit monoclonal anti-ETX antibodies and their use in various biological assays. Antibodies possessing differential specificity to ETX in particular conformations will aid in the mechanistic studies of ETX cytotoxicity, while those with ETX-neutralizing function may be useful in preventing ETX-mediated mortality.

Attenuation of acute lung injury and oxygen radical production by the 21-aminosteroid, U-78518F

1993 ◽  
Vol 74 (5) ◽  
pp. 2155-2160 ◽  
Author(s):  
T. Tanigaki ◽  
Y. Suzuki ◽  
D. Heimer ◽  
H. H. Sussman ◽  
W. G. Ross ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Phorbol Myristate Acetate ◽  
Guinea Pigs ◽  
Oxygen Radical ◽  
Myristate Acetate ◽  
E Coli ◽  
Radical Production ◽  
Oxygen Radical Production

Oxygen radicals play an important role in the mechanism of acute lung injury. The 21-aminosteroid lazaroid, U-78518F, is a potent antioxidant. We examined the effect of intravenous U-78518F on acute lung injury in septic guinea pigs over 8 h. The experimental groups (n = 6) were 1) saline control, 2) Escherichia coli (2 x 10(9)/kg i.v.), 3) pretreatment (U-78518F 5 mg/kg bolus + 1 mg.kg-1 x h-1, 15 min before E. coli injection), and 4) posttreatment (U-78518F 30 min after E. coli injection). We measured wet-to-dry weight ratio (W/D) as an index of pulmonary edema and concentration ratios of 125I-labeled albumin in lung tissue and bronchoalveolar lavage fluid compared with plasma (L/P and BAL/P, respectively) as indexes of lung protein fluxes. In septic guinea pigs, pretreatment with U-78518F attenuated W/D, L/P, and BAL/P and posttreatment attenuated W/D and BAL/P (P < 0.05 for each). Furthermore, we studied the effect of U-78518F on human neutrophil oxygen radical production (ORP) by using flow cytometry to assess intracellular ORP and lucigenin-dependent chemiluminescence to assess extracellular ORP. Neutrophils (5 x 10(5) were stimulated with 0.5 micrograms/ml of phorbol myristate acetate. With flow cytometry, we measured intracellular ORP, cross-sectional cell area, and degranulation in neutrophils. U-78518F (minimum concn 1.0 microM) decreased intracellular ORP (n = 4; P < 0.05) when the dihydrorhodamine 123 assay was used. U-78518F (minimum concn 1.0 microM) inhibited phorbol myristate acetate-induced neutrophil chemiluminescence (n = 4; P < 0.05).

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