Journal: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 9. Rac1 via actomyosin regulates the G2-M checkpoint kinase Wee1 to prevent premature mitotic entry. (A and B) CD cells were G2-synchronized using RO- 3306, and lysates were collected at the indicated time points after RO-3306 washout (“G2 release”). Lysates were immunoblotted for pH3 to monitor mitotic entry. Three repeat experiments were quantified in (B). Fold change values ± SD. Arrows highlight the first pH3 peak of the respective groups indicating mitotic entry. (C and D) G2- synchronized CD cells were immunoblotted for cleaved caspase 3 (cl-Casp3) after G2 release and quantified in (D) as fold change values ± SD (n = 3). Asterisk (*) denotes between-group significance at the corresponding time point. (E and F) CD cells were G2-synchronized, and lysates were obtained immediately upon G2 washout (G2, T0) and immunoblotted in biological duplicates. p-Cdk1 Y15: phosphorylated Cdk1 tyrosine-15. Three repeat experiments were quantified in (F). Fold change values ± SD. (G and H) Asynchronous Rac1f/f (+DMSO) and Rac1−/− [+DMSO or blebbistatin (5 μM)] CD cells were treated with cycloheximide (CHX; 100 μg/ml), and lysates were obtained at the indicated time points and immunoblotted for Wee1. Three repeat experiments are quantified in (H) as fold change ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. (I and J) Rac1f/f and Rac1−/− CD cells were G2-synchronized and treated with vehicle (DMSO) or 5 μM blebbistatin upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry. Three independent repeat experiments are quantified (for Rac1f/f, only the vehicle control is shown) in (J) as fold change values ± SD. Asterisk (*) denotes significance between Rac1−/− and Rac1f/f or blebbistatin-treated Rac1−/− at the corresponding time point. *P < 0.05.

Article Snippet: Other cell culture treatments included nocodazole (Tocris Bioscience, #1228) at 100 ng/ml or the Wee1 inhibitor Adavosertib (MK- 1775, Tocris Bioscience, #7589) at 1 μM, blebbistatin (Sigma- Aldrich, #203391) at 5 μM, or the ROCK inhibitor Y- 27632 at 1 μM.

Techniques: Control

Journal: Science advances

Article Title: Rac1 promotes kidney collecting duct repair by mechanically coupling cell morphology to mitotic entry.

doi: 10.1126/sciadv.adi7840

Figure Lengend Snippet: Fig. 10. Wee1 inhibition phenocopies Rac1 deficiency in mitosis. (A to D) Rac1f/f and Rac1−/− CD cells were G2-synchronized using RO-3306 and treated with the Wee1- specific inhibitor MK-1775 (1 μM) upon G2 release. Lysates were collected at the indicated time points and immunoblotted for pH3 to monitor mitotic entry or cleaved caspase 3 to monitor cell death. Densitometry was used to quantify fold changes ± SD of three repeat experiments in (B) and (D). Arrows in (B) highlight the first pH3 peak indicating mitotic entry. (E) F-actin (white)– and DNA (blue)–labeled Rac1f/f and Rac1−/− (+MK-1775; 1 μM) CD cell monolayers analyzed by confocal microscopy with a mitotic metaphase cell shown in the center (scale bars, 10 μm). The top row column depicts metaphase F-actin (scale bars, 5 μm) as outlined by a red continuous box in the bottom row. Images are representative of at least three experiments. (F) Circularity quantification of mitotic metaphase F-actin as shown in (E) with a minimum of 10 measurements shown per group. Bars are means ± SD. (G) Live confocal mitosis imaging of SPY650-DNA (blue)– and SPY555-actin (white)–labeled vehicle (DMSO)– or MK-1775 (1 μM)–treated Rac1f/f CD cells in vitro. The mitotic cell was manually segmented and colored in green. Scale bars, 10 μm. Sequences are representative of three repeat experiments with at least three to four mitoses analyzed per experiment. (H) Relative distribution of mitotic defects in vitro during live imaging cell division se- quences with at least 10 mitoses analyzed per group. *P < 0.05.

Article Snippet: Other cell culture treatments included nocodazole (Tocris Bioscience, #1228) at 100 ng/ml or the Wee1 inhibitor Adavosertib (MK- 1775, Tocris Bioscience, #7589) at 1 μM, blebbistatin (Sigma- Aldrich, #203391) at 5 μM, or the ROCK inhibitor Y- 27632 at 1 μM.

Techniques: Inhibition, Labeling, Confocal Microscopy, Imaging, In Vitro

Journal: Genes & Diseases

Article Title: Differences between lung adenocarcinoma and lung squamous cell carcinoma: Driver genes, therapeutic targets, and clinical efficacy

doi: 10.1016/j.gendis.2024.101374

Figure Lengend Snippet: Targeted therapies for driver genes in lung adenocarcinoma and lung squamous cell carcinoma.

Article Snippet: , ∗Adavosertib , Phase II , NCT02087176 , Docetaxel ± adavosertib , 48 , NA , NA , 9.4% , AstraZeneca.

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression prevents premature mitotic entry from S phase in Adavosertib treated cells. (A) Non treated or tetracycline treated HeLa cells were released from G1/S phase into media containing either DMSO or Adavosertib and then observed by time-lapse microscopy (1 h post G1/S release). Each line represents the fate of a single cell and forked lines indicate cell divisions. The dark grey box indicates the time when nontreated cells are expected to enter mitosis (9–11 h). (B) The flow chart depicts in vitro kinase assay and time lapse imaging protocols. Cdk1 activity in lysates from cells 4 h after G1/S release was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) In vitro Cdk1 activity (top two panels) was assessed in HeLa cells (treatments are indicated). GFP, Myt1 (band for the fusion protein is shown), and tubulin levels were analyzed by immunoblot (bottom three panels). (D) The graph shows the quantitation of average Cdk1 activity (relative to control Tet-/DMSO). Error bars represent SEM. ** and **** denote p < 0.01 and p < 0.0001 (Two-way ANOVA). Experiments were repeated at least three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Time-lapse Microscopy, In Vitro, Kinase Assay, Imaging, Activity Assay, Incubation, Western Blot, Quantitation Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression rescues cells from Adavosertib induced mitotic arrest. (A) HeLa cells were treated with 250 nM Adavosertib in the presence or absence of 2 µM tetracycline and then analyzed by time-lapse microscopy. Scale bar = 10 µm. (B) An immunoblot shows total levels of GFP, Myt1, and tubulin. (C) Time in mitosis for indicated treatments is shown. **** denotes p < 0.0001 (ANOVA). Median mitotic times are included in the table below. (D) Donut charts show the proportion of cell death for each treatment. The number of cells counted is shown within each chart. Experiments were repeated at least three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Time-lapse Microscopy, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression promotes mitotic exit in the presence of Adavosertib. (A) Nontreated and tetracycline treated HeLa cells were released from G1/S for 8 h release in fresh medium before adding DMSO or Adavosertib and then analyzed by time-lapse microscopy. Each line represents a single cell and forked lines indicate cell division. (B) The flow chart depicts the time lapse imaging protocol. (C) Donut charts indicate the proportion of cell death observed for each treatment. The number of cells counted is indicated within each donut plot. (D) Graph indicates the duration of mitosis (NEBD to anaphase/mitotic slippage). Median mitotic times are included in the table provided. Experiments were repeated at least three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Time-lapse Microscopy, Imaging

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression promotes resistance to DNA damage and cell cycle checkpoint kinase inhibitors. HeLa cells were either not treated or treated with 2 µM tetracycline for 24 h and then exposed to (A) Adavosertib (500 nM), (B) AZD6738 (1000 nM), (C) UCN-01 (1000 nM), or (D) PD166285 (500 nM) for an additional 48 h. Graphs show the average percent cell survival determined by crystal violet assay. For colony formation assays, HeLa cells with or without tetracycline were treated with (E) Adavosertib, (F) AZD6738, (G) UCN-01, or (H) PD166285 for 24 h and then replenished with fresh media to be incubated for 14 days to evaluate their clonogenic potential as shown. Error bars represent SD. Experiments were repeated three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Crystal Violet Assay, Incubation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression promotes resistance to DNA damage and cell cycle checkpoint kinase inhibitors. The table provides the IC50 values towards different cell cycle or DNA damage checkpoint kinase inhibitors calculated over 48 h either in the presence or absence of tetracycline. The percent cell survival was evaluated using crystal violet assay and the IC50 provided is in nM with a corresponding 95%CI. The experiment was repeated three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Crystal Violet Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: Myt1 overexpression inhibits mitotic entry by reducing Cdk1 activity in cells treated with checkpoint kinase inhibitors (A) HeLa cells were seeded onto coverslips in the presence or absence of tetracycline (2 µM) for 24 h. Cells were synchronized in G1-S phase by single thymidine block. Following synchronization cells were released into media containing kinase inhibitors or DMSO for 4 h. Cells were stained for DNA and PH3 and analyzed by immunofluorescence microscopy. The percentage of cells positive for PH3 is shown normalized to DMSO/Tet-. Error bars represent SEM. Experiments were repeated three times. Statistical significance was determined by 2-way ANOVA. ***, p < 0.001; **, p = 0.001. (B) Cell lysates were prepared from tetracycline inducible Myt1 expressing HeLa cells. Cells were treated or not with 2 µM of tetracycline 48 h prior to any drug treatment/control: DMSO, Adavosertib (500 nM), PD166285 (500 nM), UCN-01 (1000 nM), and AZD6738 (1000 nM) for 4 h. Cdk1 activity in lysates from cells was assessed in vitro by incubation with GST-PP1Cα (a Cdk1 substrate). Total pT320-PP1Cα peptide and GST levels were determined by immunoblot. (C) Bar graphs show the quantitation of the absolute Cdk1 activity in cells treated with various checkpoint kinase inhibitors. Error bars represent SD. Statistical analysis was done using a 2-way ANOVA. ****, p < 0.0001**, p = 0.0029. (D) Lysates were also evaluated for the levels of pY15-Cdk1, Cdk1, pS345-Chk1, Chk1, overexpressed Myt1 and Tubulin. Quantitation shows the average pS345-Chk1 and pY15-Cdk1 levels relative to their total proteins and normalized to DMSO with no tetracycline addition. Kinase assay was repeated 5 times or more. Western blotting and the immunofluorescence experiments were repeated three times.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Over Expression, Activity Assay, Blocking Assay, Staining, Immunofluorescence, Microscopy, Expressing, Control, In Vitro, Incubation, Western Blot, Quantitation Assay, Kinase Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Myt1 overexpression mediates resistance to cell cycle and DNA damage checkpoint kinase inhibitors

doi: 10.3389/fcell.2023.1270542

Figure Lengend Snippet: DNA damage and cell cycle checkpoint inhibitors and their effect on ectopic Cdk1 activation. (A) In an unperturbed cell, Cdk1 and cyclin B bind in late S-phase. Phosphorylation of Cdk1 on T14 and Y15 (depicted with red circles) by Myt1 and Wee1 during interphase inhibits Cdk1/cyclin B. Cdc25 removes these inhibitory phosphorylations and activates the Cdk1/cyclin B complexes allowing cells to enter mitosis. The high level of Wee1 activity in many cancer cells prevents their premature entry into mitosis and facilitates their survival in the presence of elevated genotoxic stress compared to normal cells. (B) Upon addition of Adavosertib (inhibition of Wee1) or PD166285 (inhibition of Wee1 and Myt1), the balance between inactive and active Cdk1/cyclin B1 complexes is shifted leading to more active Cdk1/cyclin B1 complexes. This increases the chance of premature entry into mitosis and cell death. (C) Overexpression of Myt1 in the presence of Adavosertib or PD166285 counters the drugs’ effect because phosphorylation of Cdk1 on T14 and Y15 by Myt1 increases inactive Cdk1/cyclin B complexes. This results in checkpoint arrest and rescue from cell death even in the presence of these cell cycle checkpoint kinase inhibitors. (D) Damaged DNA or replication stress can induce the activation of the DNA damage checkpoint, which downregulates Cdk1/cyclin B activity via Cdc25. ATR activates the effector kinase Chk1, which phosphorylates and inhibits the Cdc25 phosphatase family. In parallel, ATR can induce the stabilization and transcriptional activity of p53 leading to increased expression of the CDK inhibitor p21; however, this pathway is downregulated in many cancers (due to non-functional p53 status of many cancers) . (E) Upon addition of UCN-01 or AZD6738 (inhibition of Chk1 and ATR), Cdc25 inactivation in the presence of DNA damage is repressed and the balance between inactive and active Cdk1/cyclin B1 complexes is shifted towards more active Cdk1/cyclin B1 complexes. The cells are at elevated risk of premature mitotic entry. Restoration of Cdc25 activity in the presence of DNA damage is likely to have a lower effect then inhibiting Wee1 and Myt1 with Adavosertib or PD-166285. (F) Upon overexpression of Myt1 in the presence of UCN-01 or AZD6738 which leads to more inhibitory phosphorylation of Cdk1 on both T14 and Y15, the pool is reverted to more inactive Cdk1/cyclin B1 complexes even in the presence of these DNA damage checkpoint kinase inhibitors. Red arrows are Cdk1 activating pathways whereas black arrows are Cdk1 inactivating pathways. Solid line shows an active pathway, a dotted line an inhibited pathway. The p53 pathway (grey) is abrogated in most cancers. The meter schematically indicates the balance in active/inactive Cdk1 complexes in each scenario and the associated risk of premature entry into mitosis.

Article Snippet: Cells were treated with 2 μM tetracycline (FisherBiotech; 64-75-5), 2 mM thymidine (Sigma; T1895), 500 nM Adavosertib (Chemie Tek; 955365-80-7), 500 nM PD166285 (Selleckchem; S8148), 1 μM UCN-01 (Sigma-Aldrich; 112953-11-4), and 1 μM AZD6738 (provided by AstraZeneca).

Techniques: Activation Assay, Phospho-proteomics, Activity Assay, Inhibition, Over Expression, Expressing, Functional Assay

Journal: Developmental Cell

Article Title: A platform for efficient establishment and drug-response profiling of high-grade serous ovarian cancer organoids

doi: 10.1016/j.devcel.2023.04.012

Figure Lengend Snippet:

Article Snippet: Adavosertib , ChemieTek , Cat# CT-MK1775.

Techniques: Recombinant, Membrane, Plasmid Preparation, Clinical Proteomics, CellTox Assay, Gene Expression, Software