Journal: Molecules

Article Title: Design, Synthesis, and Anticancer Screening for Repurposed Pyrazolo[3,4-d]pyrimidine Derivatives on Four Mammalian Cancer Cell Lines

doi: 10.3390/molecules26102961

Figure Lengend Snippet: Two-dimensional binding mode of the test compounds ( a ) 6 , ( b ) 5 , and ( c ) 7 in the binding site of Ki67 protein (PDB ID: 2aff) using MOE software.

Article Snippet: Cells treated with IC 50 concentration of the tested compound for 48 h were labelled using the Ki67 Proliferation Kit (D3B5, Rabbit mAb Alexa Fluor ® 488 Conjugate, Cell Signaling Technology) according to manufacturer’s instructions (Cell Signaling Technology; Flow Cytometry, Methanol Permeabilization Protocol) and analysed by using BD FACS Calibur flow cytometry and Cell QuestTM software.

Techniques: Binding Assay, Software

Journal: Molecules

Article Title: Design, Synthesis, and Anticancer Screening for Repurposed Pyrazolo[3,4-d]pyrimidine Derivatives on Four Mammalian Cancer Cell Lines

doi: 10.3390/molecules26102961

Figure Lengend Snippet: ( a ) Apoptosis analysis in A549 cells following treatment of cells with compound 7 at IC 50 concentration. Flowcytometry was conducted using Alexa Fluor 488 annexin V conjugate. The percentage gating in the lower left quadrant was ( A ) 97.8 in the control untreated cells and ( B ) 97.58% in compound 7 . ( b ) Western blot analysis showing cleaved caspase-3 protein expression level. The upper panel shows (1) control, (2) compound 5 , (3) compound 6 , and (4) compound 7 at IC 50 concentration. The lower panel demonstrates B actin as loading control. ( c ) Proliferation analysis (Ki67) in A549 cells following treatment cells with compound 7 at IC 50 concentration. The analysis was done using flow cytometry and the Ki-67 FITC antibody. ( A ) shows the flow cytometric analysis of the control, whereas ( B ) shows the flow cytometric analysis of compound 7 .

Article Snippet: Cells treated with IC 50 concentration of the tested compound for 48 h were labelled using the Ki67 Proliferation Kit (D3B5, Rabbit mAb Alexa Fluor ® 488 Conjugate, Cell Signaling Technology) according to manufacturer’s instructions (Cell Signaling Technology; Flow Cytometry, Methanol Permeabilization Protocol) and analysed by using BD FACS Calibur flow cytometry and Cell QuestTM software.

Techniques: Concentration Assay, Control, Western Blot, Expressing, Flow Cytometry

Journal: Cell Reports

Article Title: Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage

doi: 10.1016/j.celrep.2020.01.074

Figure Lengend Snippet: Sustained MAPK Signaling Allows Cells to Enter Mitosis after Damage-Induced G2 Arrest (A) Representative western blot showing that CDC25C phosphorylation at T48 in synchronized MCF-7 cells is decreased in response to DNA damage and is restored by HRG. (B) Representative western blot showing the expression levels of PLK1 and CYCLIN B1 in asynchronous MCF-7 cells after DNA damage, in the absence or presence of HRG. (C) Quantification of the western blot showing an HRG-dependent increase in PLK1, even in the presence of NCS. (D) Quantification of the western blot showing an HRG-dependent increase in CYCLIN B1, even in the presence of NCS. (E) Representative western blot showing the expression of PLK1 in G2-synchronized MCF-7 cells in control and NCS-treated cells and in NCS- and HRG-treated cells. (F) Quantification of the western blot showing that HRG can restore PLK1 expressing in synchronous cells to levels similar to undamaged cells. All of the experiments represent at least 3 independent repeats. See also and .

Article Snippet: The membranes were incubated with the following primary antibodies in 5% milk blocking solution, unless otherwise indicated: mouse p53 (DO-1, Santa Cruz, 1:500), mouse phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (E10, Cell Signaling, 1:1000), rabbit p44/42 MAPK (Cell Signaling, 1:1000), mouse beta-actin (Sigma,1:10,000), mouse ATM (Monoclonal, Sigma, 1:500), rabbit phospho-ATM (Ser1981) (AbCam,1:1000, Rabbit), mouse phospho-c-Fos (Ser374) (Calbiochem, 1:500 in 5% BSA), mouse c-Fos (Santa Cruz, 1:500 in 5% BSA), rabbit phospho-cdc25C (Thr48) (Cell Signaling, 1:500 in 5% BSA), rabbit Cdc25c (C20, Santa Cruz, 1:500), mouse EGFP (JL-8, Clontech,1:1000), mouse c-Raf (Cell Signaling, 1:1000), mouse Plk1 (Invitrogen, 1:1000), mouse Cyclin B1 (Santa Cruz, 1:1000), rabbit monoclonal phospho-MAPK substrates motif PXpTP (Cell Signaling 1:1000 in 5% BSA).

Techniques: Western Blot, Phospho-proteomics, Expressing, Control

Journal: Cell Reports

Article Title: Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage

doi: 10.1016/j.celrep.2020.01.074

Figure Lengend Snippet: Cells Enter Mitosis with Unresolved DNA Damage (A) Cells stained against phosphorylated H2AX, a marker of DNA damage visible in any phase of the cell cycle, showing the presence of unresolved DNA damage in cells that have entered mitosis (marked by arrows). Accompanying bright-field images clearly show rounded mitotic cells in the field of view. There is little or no damage in mitotic cells, which have either been left untreated or treated with U0126 or HRG alone. Scale bar: 25 μm. (B) Graphical representation of the positive and negative feedback first identified by Lahav et al. (2004) , subsequently modeled with the addition of the WIP1 phosphatase role in Batchelor et al. (2011) , where ERK activity discovered in this work is exemplified with the reported MAPK-MDM2 interaction ( Gerarduzzi et al., 2016 ). (C) A simple competitive model for the DNA damage checkpoint. The pulsatile response of p53 is known to maintain DNA-damaged cells in an arrested state conducive to DNA damage repair without permanent withdrawal from the cell cycle. The pulsatile activation of the MAPK/ERK pathway after DNA damage is necessary for the maintenance of this pro-survival state. The loss of MAPK/ERK activation leads to increased p53 activity and an increased probability of permanent cell-cycle arrest, while sustained MAPK/ERK activation leads to a greater probability of checkpoint recovery from DNA damage-induced arrest mediated by CDC25C phosphorylation.

Article Snippet: The membranes were incubated with the following primary antibodies in 5% milk blocking solution, unless otherwise indicated: mouse p53 (DO-1, Santa Cruz, 1:500), mouse phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (E10, Cell Signaling, 1:1000), rabbit p44/42 MAPK (Cell Signaling, 1:1000), mouse beta-actin (Sigma,1:10,000), mouse ATM (Monoclonal, Sigma, 1:500), rabbit phospho-ATM (Ser1981) (AbCam,1:1000, Rabbit), mouse phospho-c-Fos (Ser374) (Calbiochem, 1:500 in 5% BSA), mouse c-Fos (Santa Cruz, 1:500 in 5% BSA), rabbit phospho-cdc25C (Thr48) (Cell Signaling, 1:500 in 5% BSA), rabbit Cdc25c (C20, Santa Cruz, 1:500), mouse EGFP (JL-8, Clontech,1:1000), mouse c-Raf (Cell Signaling, 1:1000), mouse Plk1 (Invitrogen, 1:1000), mouse Cyclin B1 (Santa Cruz, 1:1000), rabbit monoclonal phospho-MAPK substrates motif PXpTP (Cell Signaling 1:1000 in 5% BSA).

Techniques: Staining, Marker, Activity Assay, Activation Assay, Phospho-proteomics

Journal: Cell Reports

Article Title: Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage

doi: 10.1016/j.celrep.2020.01.074

Figure Lengend Snippet:

Article Snippet: The membranes were incubated with the following primary antibodies in 5% milk blocking solution, unless otherwise indicated: mouse p53 (DO-1, Santa Cruz, 1:500), mouse phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204) (E10, Cell Signaling, 1:1000), rabbit p44/42 MAPK (Cell Signaling, 1:1000), mouse beta-actin (Sigma,1:10,000), mouse ATM (Monoclonal, Sigma, 1:500), rabbit phospho-ATM (Ser1981) (AbCam,1:1000, Rabbit), mouse phospho-c-Fos (Ser374) (Calbiochem, 1:500 in 5% BSA), mouse c-Fos (Santa Cruz, 1:500 in 5% BSA), rabbit phospho-cdc25C (Thr48) (Cell Signaling, 1:500 in 5% BSA), rabbit Cdc25c (C20, Santa Cruz, 1:500), mouse EGFP (JL-8, Clontech,1:1000), mouse c-Raf (Cell Signaling, 1:1000), mouse Plk1 (Invitrogen, 1:1000), mouse Cyclin B1 (Santa Cruz, 1:1000), rabbit monoclonal phospho-MAPK substrates motif PXpTP (Cell Signaling 1:1000 in 5% BSA).

Techniques: Virus, Recombinant, Transfection, cDNA Synthesis, SYBR Green Assay, Staining, Software

Journal: eLife

Article Title: Leptin suppresses development of GLP-1 inputs to the paraventricular nucleus of the hypothalamus

doi: 10.7554/eLife.59857

Figure Lengend Snippet: ( A ), ( B ) Representative images of pSTAT3 immunoreactivity (green) in LepRb-Cre::tdTom mice injected with leptin or saline control at P16. ( C ), ( D ) Representative images of pSTAT3 immunoreactivity in GCG-Cre::SynTom mice injected with leptin or saline control at P16. ( E ) Percentage of LepRb and PPG neurons in the NTS that are responsive to leptin. ( F, G ) LepRb-expressing neurons are visualized by injection of AAV-EGFP virus into the NTS of a LepRb-Cre mouse. ( H ) Densely labeled terminals in the PVH from LepRb-expressing neurons in the NTS. N = 3 for each group. Abbreviations: 3V, third ventricle; AP, area postrema; DMX, dorsal motor nucleus of the vagus; NTSco, commissural nucleus of the solitary tract; NTSm, medial nucleus of the solitary tract; NTSl, lateral nucleus of the solitary tract; cc, central canal.

Article Snippet: Tissue sections were incubated for 48 hr with a rabbit anti-pSTAT3 primary antibody (1:1,000; Cell Signaling, Danvers, MA).

Techniques: Injection, Saline, Control, Expressing, Virus, Labeling

Journal: eLife

Article Title: Leptin suppresses development of GLP-1 inputs to the paraventricular nucleus of the hypothalamus

doi: 10.7554/eLife.59857

Figure Lengend Snippet:

Article Snippet: Tissue sections were incubated for 48 hr with a rabbit anti-pSTAT3 primary antibody (1:1,000; Cell Signaling, Danvers, MA).

Techniques: Recombinant, Software

Journal: The EMBO Journal

Article Title: PLK1 inhibition delays mitotic entry revealing changes to the phosphoproteome of mammalian cells early in division

doi: 10.1038/s44318-025-00400-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-Phospho-Cdc25C (Thr48) (WB) , Cell Signaling Technology , 9527.

Techniques: Recombinant, Modification, Electron Microscopy, Transfection, Bicinchoninic Acid Protein Assay, Software, Microscopy