Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Viability of AML cell lines treated with Nut-3a and/or WIP1i. ( A ) Percentage of viable MOLM-13, MV-4-11, OCI-AML3, NOMO-1, HEL and KASUMI-1 AML cells treated with increasing concentrations of single agent Nut-3a (from 0.5 to 5 μM) for 24, 48 and 72 h. ( B ) IC50 values of AML cell lines at 72 h of treatment with Nut-3a or WIP1i (NR = not reached). ( C ) Percentage of viable cells treated with increasing concentrations of single agent WIP1i (from 5 to 20 μM) for 24, 48 and 72 h. Inhibition of cell viability induced in TP53 -wt ( D ) and TP53 -mut ( E ) AML cell lines by the combination of increasing concentrations of Nut-3a (from 0.5 to 5 μM) and WIP1i (from 5 to 20 µM) at 24, 48 and 72 h. Average value and standard deviation of 3 independent experiments are shown.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Inhibition, Standard Deviation

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Apoptotic response of AML cell lines and primary cells to combined Nut-3a and WIP1i treatment. ( A ) Histograms showing the percentage of apoptotic (AnnexinV + cells) cells in TP53 -wt ( A ) and TP53 -mut cell lines ( B ) after 24 and 48 h treatment with single and combined Nut-3a and WIP1i. Average value and standard deviation of 3 independent experiments are shown. ( C ) Cell viability and ( D ) apoptotic response of TP53 -wt AML primary cells ( n = 3 and n = 6, respectively) after 24 h and 48 h treatment with single and combined Nut-3a and WIP1i treatments. ( E ) Histograms showing the percentage of dead cells in NPM1 -mut and NPM1 -wt primary AML cells ( n = 5 each) after 48 h treatment with single and combined Nut-3a and WIP1i. ( F ) Histograms showing the percentage of viable cells (normalized on vehicle-treated cells) in TP53 -mut ( n = 3) and TP53 -wt ( n = 4) primary AML cells after 48 h treatment with single and combined Nut-3a and WIP1i (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Standard Deviation

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Changes in the expression of p53-related genes induced by the treatment in TP53 -wt and TP53 -mut cells. Cells were harvested after 16 h of treatment (Nut-3a 0.5 and 5 µM; WIP1i 5 and 20 µM, for TP53 -wt and TP53 -mut cells, respectively) both for gene expression microarray and protein analyses. ( A ) Enrichment of p53 signature in MV-4-11 cells treated with the drug combination vs. vehicle (gene expression microarray). ( B ) Heatmap of MV-4-11 and NOMO-1 cells showing the significantly deregulated genes (differential expression analysis between Nut-3a+WIP1i-treated and vehicle-treated MV-4-11 cells, fold change ≥ 2, p < 0.05) belonging to the p53 signature in the analyzed models. ( C ) Protein quantification of p53-related genes in treated TP53 -wt and ( D ) TP53 -mut cells. Histograms show the average value of 3 independent experiments ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Expressing, Microarray

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Most significant enriched pathways in MV-4-11 cells upon drug treatment.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Comparison, Activity Assay

Journal: Biomedicines

Article Title: Pharmacological Inhibition of WIP1 Sensitizes Acute Myeloid Leukemia Cells to the MDM2 Inhibitor Nutlin-3a

doi: 10.3390/biomedicines9040388

Figure Lengend Snippet: Proposed mechanism of action of Nut-3a and WIP1i combined treatment in AML cells. MDM2 inhibition enhances p53-dependent response to DNA damages induced by chemotherapy agents or replicative stress. Once Nut-3a binds to MDM2, p53 is released and activated through phosphorylation. Active p53 promotes the induction of apoptosis. ( A ) WIP1 is involved in the regulation of response to Nut-3a and dephosphorylates p53. WIP1 and p53 are co-regulated by a feedback-loop. ( B ) When WIP1i is simultaneously added to Nut-3a, p53 activation is enforced, resulting in enhanced apoptosis of AML cells. The arrows represent a stimulatory signal, truncated arrows represent a inhibition signal.

Article Snippet: The following primary antibodies were used: anti-WIP1 (#SC20712) from Santa Cruz Biotechnology; anti-p53 (PAb 140, NB 200-103) from Novus Biological (Centennial, CO, USA); anti-MDM2 (D1V2Z), anti-p21 WAF1/Cip1 (12D1), all from Cell Signaling (Danvers, MA, USA); anti-β-actin from Sigma-Aldrich.

Techniques: Inhibition, Activation Assay

Journal: Cell

Article Title: A white-box machine learning approach for revealing antibiotic mechanisms of action

doi: 10.1016/j.cell.2019.04.016

Figure Lengend Snippet: (A) Overall experimental design for measuring metabolite effects on antibiotic lethality. Overnight cultures of E. coli MG1655 were inoculated into MOPS minimal medium, grown to early exponential phase, and back-diluted to OD600 = 0.1. Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 (Bochner, 2009) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added. OD600 was measured after 4 hours of incubation at 37°C and 900 rpm shaking. Antibiotic IC50s were estimated for each antibiotic-metabolite combination.

Article Snippet: Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 ( Bochner, 2009 ) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added.

Techniques: Microarray, Incubation

Journal: Cell

Article Title: A white-box machine learning approach for revealing antibiotic mechanisms of action

doi: 10.1016/j.cell.2019.04.016

Figure Lengend Snippet: Key Resources Table

Article Snippet: Cells were dispensed into Biolog phenotype microarray plates (PMs) 1–4 ( Bochner, 2009 ) with different concentrations of ampicillin (AMP), ciprofloxacin (CIP) or gentamicin (GENT) added.

Techniques: Virus, Recombinant, Microarray, Software, Combined Bisulfite Restriction Analysis Assay

Journal: Cancers

Article Title: A Systemic and Integrated Analysis of p63-Driven Regulatory Networks in Mouse Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers15020446

Figure Lengend Snippet: Figure 11. Immunohistochemical staining in HNSCC tumor microarray tissues. Staining of p63 (A), COTL1 (B), and K14 (C) across normal, malignant tumor stage II, and malignant tumor stage III tissues at 10× magnification.

Article Snippet: After blocking in 5% milk, the membranes were incubated first in primary antibodies against p63 (4A4, 1:20,000), COTL1 (Proteintech, 1:10,000), K14 (a gift from Dr. RoseAnne Romano) [29], Vimentin (CST, 1:5000), MMP9 (Proteintech, 1:10,000), Fibronectin (SinoBiological, 1:5000), ITGB4 (Proteintech, 1:10,000), E-cadherin (CST, 1:5000), and K6 (a gift from Dr. Julie Segre), then with horseradish peroxidase-conjugated secondary antibodies corresponding to the host of the primary antibody, and then washed in Trisbuffered saline with 0.05% Tween-20.

Techniques: Immunohistochemical staining, Staining, Microarray

Journal: Cell

Article Title: Cancer-germline antigen expression discriminates clinical outcome to CTLA-4 blockade

doi: 10.1016/j.cell.2018.03.026

Figure Lengend Snippet: (A) 14-3-3ζ is downregulated in melanoma tumors with high CRMA expression. (B) In vitro screen for MAGE-TRIM28 ubiquitination substrates identifies HMGB1 (p=0.04). AMPKα1 was used as a positive control (Pineda et al., 2015). (C) Immunofluorescence staining (IF) for MAGE-A and HMGB1 in a melanoma tissue microarray (TMA) shows a negative association between the two proteins in individual cells. (D) Examples from the TMA of mutually exclusive expression of MAGE-A and HMGB1 in cells from the same tumor. Magnification x200. (E) A higher proportion of MAGE− tumors are positive for LC3B, a marker of autophagy, by IHC staining compared to MAGE+ tumors in the melanoma TMA (p=0.004) (top). Examples of LC3B+ and LC3B− tumors, magnification x400 (bottom). (F) Immunofluorescence staining for MAGE-A and HMGB1 shows mutual exclusion in five patient samples from the discovery cohort in addition to a human xenograft melanoma. Magnification x400. (G) MAGE-TRIM28 complex drives primary resistance to CTLA-4 blockade by degradation of regulators of autophagy. See also Figure S3.

Article Snippet: Immunohistochemistry for LC3B (Novus Biologicals, Zug, Switzerland, rabbit polyclonal, #NB600-1384, 1:4’000, tris buffer, 95°C, 30 minutes) was performed using an automated immunostainer Leica Bond RX (Leica Biosystems, Heerbrugg, Switzerland) and the appropriate positive and negative controls as described before ( Schlafli et al., 2015 ).

Techniques: Expressing, In Vitro, Ubiquitin Proteomics, Positive Control, Immunofluorescence, Staining, Microarray, Marker, Immunohistochemistry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Maternal Wnt/STOP signaling promotes cell division during early Xenopus embryogenesis

doi: 10.1073/pnas.1423533112

Figure Lengend Snippet: Gsk3 negatively regulates mitotic effectors in Xenopus extracts. (A) Scheme of the protein microarray experiment to assess Gsk3-dependent polyubiquitination. Interphase egg extracts were supplemented with ubiquitin, pretreated with LiCl or NaCl (Control), and then applied on protein arrays containing ∼9,000 proteins. Polyubiquitination of arrayed proteins (PolyUb) was monitored using anti-polyubiquitin antibodies and immunofluorescence detection using an array scanner. Signal intensities were transformed and are displayed as heat-map. (B) Distribution plot of polyubiquitination changes of 3,873 in vitro polyubiquitinated proteins after Gsk3 inhibition by LiCl (Log2[Control/LiCl]). Arrays were treated with 30 mM LiCl or 30 mM NaCl (Control). LiCl reduced polyubiquitination of 864 proteins >1.8-fold. Some candidates, associated with cell cycle progression and selected for further validation are indicated in the plot. (C) Cluster of mitotic associated proteins that are significantly regulated by LiCl in B. The functional network was determined using String 9.1 and color-coded based on the fold change in the microarray (Dataset S1). (D) Western blots of indicated proteins from Xenopus eggs (naturally arrested in metaphase II) and released into interphase with the Ca2+ ionophore A23187 for 25 min, treated with LiCl, or mock treated (NaCl). β-cat, β-catenin; cycE, cyclin E; Interph., interphase; M. II, metaphase II; pH3, phosphorylated histone 3. (E) Western blots of stage VI oocytes in Prophase I and Progesterone-matured (Prog.) oocytes in metaphase II. Oocytes were injected (Inj.) with Morpholinos (MO) or mRNA as indicated. Co, control; dnWnt11, dominant negative Wnt11; pH3, phosphorylated histone 3; PPL, preprolactin (negative control).

Article Snippet: Extracts (0.6 mL) were prewarmed to room temperature, treated with 120 mM LiCl or NaCl (Control), and incubated on the microarrays under coverslips at room temperature for 1 h. Microarrays were washed with TBST and incubated overnight at 4 °C with anti-polyubiquitin antibody FK-1 (4 μg/mL) (ENZO Life Sciences) diluted in TBST, washed again, and incubated with Alexa Fluor 680 anti-mouse antibody (Invitrogen) for 4 h. The microarrays were washed three times in TBST and two times in water, dried, and scanned in an Odyssey scanner (LI-COR) using the following parameters: channel 700; grid = 8 × 3, 21.17; intensity = 5; off-set = 0.

Techniques: Microarray, Immunofluorescence, Transformation Assay, In Vitro, Inhibition, Functional Assay, Western Blot, Injection, Dominant Negative Mutation, Negative Control