Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Cytarabine dose-dependently decreases cell number and induces differentiation. ( A ) DNA damage pathway activation by pyrimidine synthesis inhibitors and cytarabine. ( B ) U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM). The number of viable cells and the expression of differentiation markers were determined after 72 h. Mean fluorescence intensity (MFI) of CD11b and CD64 was calculated as described under “ ” section. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t -test) compared with control (ctrl). ( C ) Representative histograms (out of three independent flow cytometric analyses shown in B) with black line representing isotypic control and red line representing the expression of CD11b. ( D ) Morphological analysis of U937 cells treated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10 and 100 nM). May-Grünwald-Giemsa stained cytospin preparations (100 × magnification). (E) Respiratory burst in U937 cells treated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM) for 72 h. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Activation Assay, Incubation, Expressing, Fluorescence, Control, Staining

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Cytarabine induces cell cycle arrest and activates Chk1. U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM). ( A ) Representative histograms of propidium-labelled cells from three independent experiments analyzed by flow cytometry. ( B, C ) Pan-caspase inhibitor Z-VAD-FMK (10 μM) was added 30 min before the addition of agents. ( B ) The representative dot plots of cells stained with annexin V-FITC/PI and analyzed by flow cytometry. ( C ) The percentage of annexin V-FITC-positive cells, the number of viable cells and the expression of differentiation markers were determined after 72 h. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl). ( D ) U937 cells were incubated with AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (10, 100, 1000 nM) for 48 h (upper panels) or 72 h (lower panels). Total cell lysates were isolated after 48 and 72 h and analyzed by Western blotting for the level of Ser-345-phosphorylated Chk1, total Chk1, Tyr-15-phosphorylated CDC2 and total CDC2. Representative immunoblots from three independent experiments are shown.

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Incubation, Flow Cytometry, Staining, Expressing, Control, Isolation, Western Blot

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Pharmacological inhibition of ATR/Chk1 pathway prevents differentiation and cell cycle arrest. U937 cells were grown in the presence of increasing concentrations of AraC (10, 100 nM). Torin2 (100 nM), VE-821 (10 µM) or vehicle (DMSO) were added 30 min before the addition of AraC. (A) Total cell lysates were isolated after 48 h and analyzed by Western blotting for the level of Ser-345-phosphorylated Chk1, total Chk1, Tyr-15-phosphorylated CDC2 and total CDC2. Representative immunoblots from three independent experiments are shown. ( B – C ) The number of viable cells, the expression of differentiation markers and the cell cycle progression were determined for Torin2 (B) and VE-821 (C) pre-treated cells as described under “ ” section. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t -test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Inhibition, Isolation, Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis

doi: 10.1038/s41598-022-15520-z

Figure Lengend Snippet: Down-regulation of Chk1 reduces the effects of cytarabine on the expression of differentiation markers and S-phase arrest. U937 cells were transfected with siRNA against CHK1, and respective nontargeting siRNA was used as a negative control. AICAr (AIC) (0.2 mM), brequinar (Bq) (0.5 µM) and AraC (100 nM) were added 24 h after transfection. ( A ) Total cell lysates were isolated 3 or 48 h after the addition of agents and analyzed by Western blotting for the level of Chk1. Western blot analyses are shown for each of the three independent experiments. ( B ) The number of viable cells, the expression of differentiation markers and the cell cycle progression were analyzed by flow cytometry 72 h after addition of agents. ( C ) Representative histograms of propidium-labeled cells analyzed by flow cytometry. ( D ) Percentage of cells in G 0 /G 1 , S, and G 2 /M-phases of the cell cycle. Results are mean ± S.E. (error bars) of at least three independent experiments. *, p < 0.05 (Student's t-test) compared with control (ctrl).

Article Snippet: Human AML cell lines U937 (ECACC Cat# 85,011,440) and THP-1 (DSMZ Cat# ACC-16) were purchased from European Collection of Animal Cell Cultures (Porton, Salisbury, UK) or Leibniz Institute-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively.

Techniques: Expressing, Transfection, Negative Control, Isolation, Western Blot, Flow Cytometry, Labeling, Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

doi: 10.1167/iovs.18-24132

Figure Lengend Snippet: βA3/A1-crystallin is differentially expressed in nonpolarized and polarized human RPE cells. (A, B) Polarization of RPE cells results in a highly significant increase in the mRNA and protein expression of βA3/A1-crystallin, compared to nonpolarized RPE cells. All values are represented as mean ± S.D. **P < 0.01 (Mann-Whitney U test), n = 4. (C) The distribution of βA3/A1-crystallin in polarized cultured human RPE cells as seen by confocal microscopy (upper) indicates that βA3/A1-crystallin is predominantly localized to the apical region of RPE cells. This was confirmed by a confocal cross-section image (lower). Scale bar: 20 μm for nonpolarized cells, 60 μm for polarized cells.

Article Snippet: Western blots were performed as described previously., The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized, E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), Cortactin (Cat# ab33333; Abcam), phospho(Y421)-cortactin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA).

Techniques: Expressing, MANN-WHITNEY, Cell Culture, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

doi: 10.1167/iovs.18-24132

Figure Lengend Snippet: Cryba1 is involved in the onset of EMT in the mouse RPE cells. (A, B) The mRNA levels of Snai1 and Snai2 were increased in the RPE from 5-month-old Cryba1 cKO mice compared to age-matched Cryba1 fl/fl controls, as assessed by qRT-PCR. Hprt was used as internal reference gene. (C) Western blotting revealed reduced E-cadherin and increased vimentin with no noticeable expression of βA3/A1-crystallin in 5-month-old Cryba1 cKO RPE cells compared to age-matched Cryba1 fl/fl RPE cells. Actin was used as internal control. The graphs show mean ± SD and *P < 0.05 (Mann-Whitney U test), n = 4.

Article Snippet: Western blots were performed as described previously., The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized, E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), Cortactin (Cat# ab33333; Abcam), phospho(Y421)-cortactin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, MANN-WHITNEY

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

doi: 10.1167/iovs.18-24132

Figure Lengend Snippet: Knockdown of Cryba1 triggers EMT-like phenotype in ocular melanoma cells. (A) Knockdown of Cryba1 (OCM3 cells transfected with Cryba1 siRNA) resulted in decrease in E-cadherin expression with an increase in vimentin expression, while overexpression of βA3/A1-crystallin (OCM3 cells transfected with Cryba1 pcDNA) did not cause any changes in the expression of these proteins in OCM3 cells. Representative Western blot images are shown. The graphs show mean ± S.D; *P < 0.05, **P < 0.001 (Mann-Whitney U test), n = 4. (B) Wound healing assay revealed that OCM3 cells transfected with Cryba1 siRNA showed increased wound healing after 24 hours of exposure compared to OCM3 cells transfected with Cryba1 pcDNA. The migration rate of control cells was taken as 1% and healing rate of other plates were compared with respect to control cells. (C) Increased cell migration is evident after 24 hours in Cryba1 siRNA transfected cells compared to cells transfected with Cryba1 pcDNA, suggesting the role of βA3/A1-crystallin in inhibiting ocular melanoma cell migration. The migration strips are divided into 5 units (each unit = 100 μm), black arrows indicate the cluster of cells. The migration was quantified under microscope and replicated thrice for each group. The data are represented as ‘units' migrated. All values are mean + SD and **P < 0.01 indicates significant change compared to Cryba1 siRNA transfected OCM3 cells at 0 hours.

Article Snippet: Western blots were performed as described previously., The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized, E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), Cortactin (Cat# ab33333; Abcam), phospho(Y421)-cortactin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA).

Techniques: Transfection, Expressing, Over Expression, Western Blot, MANN-WHITNEY, Wound Healing Assay, Migration, Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: A Role for βA3/A1-Crystallin in Type 2 EMT of RPE Cells Occurring in Dry Age-Related Macular Degeneration

doi: 10.1167/iovs.18-24132

Figure Lengend Snippet: Association between βA3/A1-crystallin and cortactin regulates cell migration. (A) Pull-down assay using antibody to βA3/A1-crystallin demonstrates that cortactin and βA3/A1-crystallin interact in OCM3 lysates. (B) Western blot analysis revealed that phosphorylated (Y421) cortactin is present at a higher level in cultured OCM3 cells transfected with Cryba1 siRNA compared to untreated control. (C) Pull down assay followed by Western blot showed increased cortactin and βA3/A1-crystallin association in the RPE+Choroid from WT (control) compared to Nuc1 (mutation in Cryba1 gene, forming nonfunctional βA3/A1-crystallin protein) rats. (D) Western blotting demonstrated increased expression of p-cortactin Y421 in RPE+Choroid from Nuc1 rat compared to WT, with no change in the total cortactin level. These results suggested that mutated βA3/A1-crystallin loses its ability to bind cortactin and the loss of this association triggers phosphorylation of cortactin at Y421 resulting in actin disassembly and cell migration. All values are mean ± S.D and are evaluated from three independent experiments. *Denotes significant change (P < 0.05) with respect to control OCM3 cells or RPE cell lysates from WT rats (Mann-Whitney U test), n = 4.

Article Snippet: Western blots were performed as described previously., The primary antibodies used in this study were βA3/A1-crystallin (Cat# sc-22398; Santa Cruz Biotechnology and Cat# ab151722; Abcam, Cambridge, MA, USA) as well as a polyclonal antibody developed in our laboratory and previously characterized, E-cadherin (Cat# 610181, BD Biosciences, Franklin Lakes, NJ, USA), Vimentin (Cat# 550513; BD Biosciences and Cat# MA5-11883; Thermo Fisher Scientific, Waltham, MA, USA), β-Catenin (Cat# ab16051; Abcam), Cortactin (Cat# ab33333; Abcam), phospho(Y421)-cortactin (Cat# 4569S; Cell Signaling Technology, Danvers, MA, USA), Snail1 (Cat# 14-9859-82; Invitrogen, Carlsbad, CA, USA), and Actin (Cat# A2066; Sigma Aldrich Corp., St. Louis, MO, USA).

Techniques: Migration, Pull Down Assay, Western Blot, Cell Culture, Transfection, Mutagenesis, Expressing, MANN-WHITNEY

Journal: Molecules

Article Title: The Activity of Red Nigerian Propolis and Some of Its Components against Trypanosoma brucei and Trypanosoma congolense

doi: 10.3390/molecules28020622

Figure Lengend Snippet: EC 50 of cytotoxicity of red Nigerian propolis, its fractions, and isolated compounds against U937 cells (AVG ± SEM; n = 3).

Article Snippet: U937 cells (European Collection of Cell Cultures Cat. No. 85011440, supplied by Sigma Aldrich, Dorset, UK) were cultured as described previously [ ].

Techniques: Isolation

Journal: Chembiochem

Article Title: Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1

doi: 10.1002/cbic.201800769

Figure Lengend Snippet: Schematic representation of functional screening with a CRISPR sgRNA library to search for the genes involved in the cytotoxicity of malformin A1. A) Structure of malformin A1 (MA1). B) Cytotoxic effects of MA1 in U937 cells. U937 cells were treated with 1 μ m MA1 for the periods indicated. The cytotoxicity was evaluated by 3‐(4,5‐ di methyl thiazol ‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Data are presented as means±SDs ( n =3). C) Scheme showing MA1 resistance screen in U937 cells.

Article Snippet: U937 cells (JCRB, Japan, cat# IFO50 038) were cultured in RPMI‐1640 containing fetal bovine serum (FBS, 10 %), penicillin (100 units mL −1 ), and streptomycin (100 μg mL −1 ).

Techniques: Functional Assay, CRISPR, MTT Assay

Journal: Chembiochem

Article Title: Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1

doi: 10.1002/cbic.201800769

Figure Lengend Snippet: Lentiviral CRISPR screening to identify the gene involves in the cytotoxicity of malformin A1. A) Growth of GeCKO‐transduced U937 cells. Transduced cells were treated with vehicle (DMSO) or 1 μ m MA1 for 14 d. B) Box‐whisker plot showing the distribution of sgRNA frequencies at each time point of MA1 treatment. The box extends from the first to the third quartile, with the whiskers denoting 1.5 times the interquartile range. Statistical analyses were performed by using one‐way ANOVA with the Tukey post‐hoc test. C) Scatter plots between MA1‐treated and untreated populations. The enrichment of SQLE sgRNAs was observed after MA1 treatment. SQLE sgRNAs are highlighted in red. D) sgRNA ranking determined from the difference in abundance between MA1‐treated and untreated populations. SQLE sgRNAs are highlighted in red. E) Gene hit identification by comparing differential abundances of all sgRNAs targeting a gene with differential abundances of nontargeting sgRNAs in a one‐sided Kolmogorov–Smirnov test. F) Twelve sgRNAs overlapped between the top 100 most enriched sgRNAs 7 and 14 d after MA1 treatment. G) Gene ontology in twelve overlapped sgRNAs.

Article Snippet: U937 cells (JCRB, Japan, cat# IFO50 038) were cultured in RPMI‐1640 containing fetal bovine serum (FBS, 10 %), penicillin (100 units mL −1 ), and streptomycin (100 μg mL −1 ).

Techniques: CRISPR, Whisker Assay

Journal: Chembiochem

Article Title: Genome‐Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1

doi: 10.1002/cbic.201800769

Figure Lengend Snippet: SQLE is involved in the cytotoxicity of malformin A1. A) Western blot analysis of SQLE‐depleted cells. Membrane fractions of sgRNA/Cas9‐transduced U937 cells were applied. Squalene synthase (SQS) was used as a loading control. B) Cell viability test of SQLE‐depleted cells. Control sgRNA‐ or SQLE sgRNA‐transduced U937 cells were treated with vehicle (DMSO) or 1 μ m MA1. The viable cell numbers were counted by trypan blue exclusion at the indicated time points. C) Effect of tolnaftate on MA1 cytotoxicity. U937 cells were treated with vehicle (DMSO) or with 1 μ m MA1 and/or 1 μ m tolnaftate (TNF). The viable cell numbers were counted by Trypan Blue exclusion at the indicated time points.

Article Snippet: U937 cells (JCRB, Japan, cat# IFO50 038) were cultured in RPMI‐1640 containing fetal bovine serum (FBS, 10 %), penicillin (100 units mL −1 ), and streptomycin (100 μg mL −1 ).

Techniques: Western Blot, Membrane, Control