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Optics Express Jan 2020Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is...
Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is based on decoding temporally stretched spectral interferograms that carry the spatial profiles of cells flowing on a microfluidic chip. However, the utility of OTS-QPI is troubled by difficulties in phase retrieval from the high-frequency region of the temporal interferograms, such as phase-unwrapping errors, high instrumentation cost, and large data volume. To overcome these difficulties, we propose and experimentally demonstrate frequency-shifted OTS-QPI by bringing the phase information to the baseband region. Furthermore, to show its boosted utility, we use it to demonstrate image-based classification of leukemia cells with high accuracy over 96% and evaluation of drug-treated leukemia cells via deep learning.
Topics: Antineoplastic Agents; HL-60 Cells; Humans; Imaging, Three-Dimensional; K562 Cells; Leukemia; Microfluidics; Optics and Photonics; Time Factors
PubMed: 32118978
DOI: 10.1364/OE.380679 -
Journal of Immunological Methods Oct 2021Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human... (Comparative Study)
Comparative Study
BACKGROUND AND AIMS
Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy.
METHODS
Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres.
RESULTS AND DISCUSSION
All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research.
CONCLUSION
The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.
Topics: Adenine; Autophagy; Autophagy-Related Proteins; Cell Differentiation; Cytokines; Dendritic Cells; Flow Cytometry; Genotype; HL-60 Cells; Humans; Macrolides; Microscopy, Fluorescence; Monocytes; Phenotype; Polymorphism, Single Nucleotide; Sirolimus; THP-1 Cells; U937 Cells
PubMed: 34324891
DOI: 10.1016/j.jim.2021.113106 -
Cancer Biology & Therapy Jul 2020Acute myeloid leukemia (AML) is a prevalent class of blood disease with a high occurrence rate and relapse rate. The role of dysregulated microRNAs (miRNAs) in AML is...
Acute myeloid leukemia (AML) is a prevalent class of blood disease with a high occurrence rate and relapse rate. The role of dysregulated microRNAs (miRNAs) in AML is emerging. MiR-4260 was identified to be a carcinogenic miRNA in colorectal cancer, but never has it been reported in AML. We aimed to study the function and mechanism of miR-4260 in AML. The miR-4260 level was higher in AML cell lines than the normal cell lines. Inhibition of miR-4260 hindered proliferation and increased apoptosis of AML cells. Mechanistically, long intergenic non-protein coding RNA 1128 (LINC01128) competed with nuclear receptor subfamily 3 group C member 2 (NR3C2) for miR-4260 so as to upregulate NR3C2. We identified the reduced levels of LINC01128 and NR3C2 in AML and it was suggested through rescue assays that LINC01128 repressed AML progression through regulating miR-4260/NR3C2 axis. In conclusion, we firstly uncovered that LINC01128 resisted acute myeloid leukemia through regulating miR-4260/NR3C2, providing novel clues for the treatment improvement of AML.
Topics: Apoptosis; HEK293 Cells; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; RNA, Long Noncoding; Receptors, Mineralocorticoid; THP-1 Cells; Transfection; U937 Cells; Up-Regulation
PubMed: 32338183
DOI: 10.1080/15384047.2020.1740054 -
Nature Communications May 2020Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master...
Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.
Topics: Algorithms; Dimethylpolysiloxanes; Elastic Modulus; Equipment Design; Flow Cytometry; HEK293 Cells; HL-60 Cells; Humans; Hydrodynamics; Microfluidic Analytical Techniques; Microfluidics; Models, Theoretical; Polyethylene Glycols; Rheology; Spheroids, Cellular
PubMed: 32366850
DOI: 10.1038/s41467-020-15813-9 -
Nature Communications Dec 2021The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial...
The molecular nanoscale organization of the surfaceome is a fundamental regulator of cellular signaling in health and disease. Technologies for mapping the spatial relationships of cell surface receptors and their extracellular signaling synapses would unlock theranostic opportunities to target protein communities and the possibility to engineer extracellular signaling. Here, we develop an optoproteomic technology termed LUX-MS that enables the targeted elucidation of acute protein interactions on and in between living cells using light-controlled singlet oxygen generators (SOG). By using SOG-coupled antibodies, small molecule drugs, biologics and intact viral particles, we demonstrate the ability of LUX-MS to decode ligand receptor interactions across organisms and to discover surfaceome receptor nanoscale organization with direct implications for drug action. Furthermore, by coupling SOG to antigens we achieved light-controlled molecular mapping of intercellular signaling within functional immune synapses between antigen-presenting cells and CD8 T cells providing insights into T cell activation with spatiotemporal specificity. LUX-MS based decoding of surfaceome signaling architectures thereby provides a molecular framework for the rational development of theranostic strategies.
Topics: Antibodies; Antigen-Presenting Cells; B-Lymphocytes; Biological Products; CD8-Positive T-Lymphocytes; Cell Communication; Cell Line, Tumor; Chromatography, Liquid; Gene Expression; HL-60 Cells; Humans; Immunological Synapses; Ligands; Light; Lymphocyte Activation; Optogenetics; Precision Medicine; Protein Binding; Proteomics; Receptors, Cell Surface; Signal Transduction; Singlet Oxygen; Small Molecule Libraries; Tandem Mass Spectrometry; Virion
PubMed: 34857745
DOI: 10.1038/s41467-021-27280-x -
IUBMB Life Jan 2021A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out...
A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out which settings should be activated or inhibited in autophagy pathway to prevail drug resistance and also to improve current treatment options in leukemia. Therefore, this study aimed to compare the effects of well-known inhibitors of autophagy (as 3-MA, BafA1, and HCQ) in leukemia KG-1 and HL-60 cells exposed to arsenic trioxide (ATO) and/or all-trans retinoic acid (ATRA). Cell proliferation and cytotoxicity of cells were examined by MTT assay. Autophagy was studied by evaluating the development of acidic vesicular organelles, and the autophagosomes formation was investigated by acridine orange staining and transmission electron microscopy. Moreover, the gene and protein expressions levels of autophagy markers (ATGs, p62/SQSTM1, and LC-3B) were also performed by qPCR and western blotting, respectively. The rate of apoptosis and cell cycle were evaluated using flow cytometry. We compared the cytotoxic and apoptotic effects of ATO and/or ATRA in both cell lines and demonstrated that some autophagy markers upregulated in this context. Also, it was shown that autophagy blockers HCQ and/or BafA1 could potentiate the cytotoxic effects of ATO/ATRA, which were more pronounced in KG-1 cells compared to HL-60 cell line. This study showed the involvement of autophagy during the treatment of KG-1 and HL-60 cells by ATO/ATRA. This study proposed that therapy of ATO/ATRA in combination with HCQ can be considered as a more effective strategy for targeting leukemic KG-1 cells.
Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Autophagy; Cell Proliferation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured
PubMed: 33205598
DOI: 10.1002/iub.2411 -
Molecular Medicine Reports Jan 2022Corilagin is the primary active component of the plant and has significant anti‑cancer properties. However, the biological effects and mechanisms of corilagin on...
Corilagin is the primary active component of the plant and has significant anti‑cancer properties. However, the biological effects and mechanisms of corilagin on acute myeloid leukemia (AML) have not been clarified. The Cell Counting Kit‑8 and Carboxyfluorescein Diacetate Succinimidyl Ester assay results showed that corilagin significantly inhibited proliferation of the AML cell line HL‑60 in a time‑ and dose‑dependent manner. Western blotting and flow cytometry analysis were performed to determine the levels of apoptosis in HL‑60 cells. The protein levels of cleaved caspase‑3 and Bak were upregulated, while Bcl‑xl was downregulated in cells treated with corilagin. The percentage of early‑ and late‑stage apoptotic cells increased following corilagin treatment in a dose‑dependent manner, indicating that the intrinsic mitochondrial apoptosis pathway was activated by corilagin. Simultaneously, western blotting and immunofluorescence results revealed that autophagy was suppressed; this was accompanied by a decrease in light chain 3‑II (LC3‑II) conversion and autophagosomes. MicroRNA (miRNA/miR) profile analysis showed that corilagin elevated the expression of the tumor suppressor miR‑451, while the mRNA and protein levels of high mobility group protein B1 (HMGB1), the target of miR‑451, decreased following exposure to corilagin. Knockdown of miR‑451 decreased the downregulation of HMGB1 caused by corilagin, indicating negative regulation of HMGB1 by miR‑451 during corilagin treatment. Furthermore, knockdown of miR‑451 also attenuated corilagin‑induced proliferation inhibition of HL‑60 cells, implying that miR‑451 was essential for the proliferation inhibitory effect of corilagin. In conclusion, these results indicated that corilagin induced apoptosis and inhibited autophagy in HL‑60 cells by regulating the miR‑451/HMGB1 axis, and corilagin may be a novel therapeutic drug for the treatment of AML.
Topics: Apoptosis; Autophagy; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glucosides; HL-60 Cells; HMGB1 Protein; Humans; Hydrolyzable Tannins; Leukemia, Myeloid, Acute; MicroRNAs
PubMed: 34850958
DOI: 10.3892/mmr.2021.12550 -
Blood Mar 2021Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with...
Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with Shwachman-Diamond syndrome (SDS). Here, we investigate srp54 knockout zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish experience embryonic lethality and display multisystemic developmental defects along with severe neutropenia. In contrast, srp54+/- zebrafish are viable, fertile, and show only mild neutropenia. Interestingly, injection of human SRP54 messenger RNAs (mRNAs) that carry mutations observed in patients (T115A, T117Δ, and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the various phenotypes observed in patients may be a result of mutation-specific dominant-negative effects on the functionality of the residual wild-type SRP54 protein. Overexpression of mutated SRP54 also consistently induced neutropenia in wild-type fish and impaired the granulocytic maturation of human promyelocytic HL-60 cells and healthy cord blood-derived CD34+ hematopoietic stem and progenitor cells. Mechanistically, srp54-mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Moreover, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced, xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to the loss of SRP54. The heterogenic phenotypes observed in patients that range from mild CN to SDS-like disease may be the result of different dominant-negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.
Topics: Animals; Congenital Bone Marrow Failure Syndromes; Disease Models, Animal; Gene Deletion; Gene Expression Regulation, Developmental; Gene Knockout Techniques; HL-60 Cells; Humans; Models, Molecular; Mutation; Neutropenia; RNA Splicing; RNA, Messenger; Signal Recognition Particle; X-Box Binding Protein 1; Zebrafish; Zebrafish Proteins
PubMed: 33227812
DOI: 10.1182/blood.2020008115 -
European Review For Medical and... Aug 2019To explore the regulatory role of PLZF in the malignant phenotype of non-APL acute myeloid leukemia (AML) and its underlying mechanism.
OBJECTIVE
To explore the regulatory role of PLZF in the malignant phenotype of non-APL acute myeloid leukemia (AML) and its underlying mechanism.
MATERIALS AND METHODS
The expression level of PLZF in AML cell lines KG-1a, HL-60, OCI-AML3, THP-1 and K562 was detected by quantitative Polymerase Chain Reaction (qPCR) and Western blot, respectively. Subsequently, THP-1 cells were divided into mock group (no treatment), scramble group (transfection with scramble shRNA) and shPLZF group (transfection with shPLZF). THP-1 cell line stably expressing shPLZF was constructed, followed by determination of its transfection efficiency by qPCR and Western blot, respectively. The proliferation and colony formation of THP-1 cells were accessed using CCK-8 (cell counting kit-8) assay and colony formation assay, respectively. The apoptotic rate in THP-1 cells was determined using flow cytometry. Protein levels of apoptosis-related genes in THP-1 cells were detected by Western blot. Finally, protein levels of AKT, Foxo3a, pAKT and pFoxo3a were detected by Western blot as well.
RESULTS
Both mRNA and protein levels of PLZF were relatively high in THP-1 cells, and were selected for the following experiments. After construction of THP-1 cell line stably expressing shPLZF, proliferative rate and colony formation abilities increased in the shPLZF group compared with the mock group and the scramble group. We found a decreased apoptotic rate, downregulated Bax and upregulated Bcl-2 in the shPLZF group than those of the mock group and scramble group. Phosphorylation levels of AKT and Foxo3a increased after interference with PLZF, whereas no significant changes in total levels of AKT and Foxo3a were observed.
CONCLUSIONS
PLZF inhibits the malignant phenotype of AML by regulating the AKT/Foxo3a pathway.
Topics: Apoptosis; Cell Proliferation; Forkhead Box Protein O3; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Promyelocytic Leukemia Zinc Finger Protein; Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 31378879
DOI: 10.26355/eurrev_201908_18522 -
Oxidative Medicine and Cellular... 2021Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to...
Decitabine (DAC) is a well-known DNA methyltransferase inhibitor, which has been widely used for the treatment of acute myeloid leukemia (AML). However, in addition to hypomethylation, DAC in AML is also involved in cell metabolism, apoptosis, and immunity. The TP53-induced glycolysis and apoptosis regulator (TIGAR) functions to inhabit glycolysis and protect cancer cells from reactive oxygen species- (ROS-) associated apoptosis. Our previous study revealed that TIGAR is highly expressed in myeloid leukemia cell lines and AML primary cells and associated with poor prognosis in adult patients with cytogenetically normal AML. In the present study, it was found that in a time- and concentration-dependent manner, DAC downregulates the TIGAR expression, induces ROS production, and promotes apoptosis in HL-60 and K562 cells. However, blocking the glycolytic pathway partially reversed the combined effects of DAC and TIGAR knockdown on apoptosis, ROS production, and cell cycle arrest, indicating that DAC induced apoptosis through the glycolytic pathway. Furthermore, TIGAR also has a negative impact on autophagy, while DAC treatment upregulates autophagy-related proteins LC3, Beclin-1, ATG3, and ATG-5, downregulates p62, and promotes the formation of autophagosomes, indicating that DAC may activate autophagy by downregulating TIGAR. Taken together, DAC plays an unmethylated role in inducing apoptosis and activating autophagy in myeloid leukemia by downregulating TIGAR.
Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Decitabine; Down-Regulation; Gene Expression Regulation, Leukemic; Glycolysis; HL-60 Cells; Humans; K562 Cells; Leukemia, Myeloid; Phosphoric Monoester Hydrolases; Reactive Oxygen Species
PubMed: 33532040
DOI: 10.1155/2021/8877460