Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Aneuploidy correlates and H2AX expression correlates with DDR pathway activity in gastric cancer (A) Correlation plot between aneuploidy score and CIN25 score (DNA damage checkpoint expression) for the CCLE gastric cancer cell lines. R squared value and p value calculated by simple linear correlation analysis. (B) H2AX expression (log2(TPM+1)) between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in the BROAD institute PRISM repurposing drug screen dataset; difference between the H2AX expression is represented as the mean ± S.D.; p value calculated by unpaired t test. (C) Ploidy abnormalities scores and p53 status for the five gastric cancer cell lines. (D) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in untreated five gastric cancer cell lines and RPE1 cells (non-neoplastic cell line) representing intrinsic replication stress/double-stranded DNA breaks (DSBs); nucleus stained with DAPI (blue); Scale bar: 50 μm. (E) Quantification of pH2AX and pKAP1 in indicated cell lines expressed as mean fluorescence intensity per cell (MFI) ± S.D from the following number of cells- RPE1-718, AGS- 2080, HGC27, 1031, NUGC3- 2119, GSU- 2092, KE39- 577. (F) Quantification of pH2AX staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (red). Data expressed as mean fluorescence intensity (MFI) of pH2AX signal per cell ± S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (G) Quantification of pKAP1 staining in prexasertib (Chk1/2) dose-dependent induced replication stress in cell lines with “intrinsic low-replication stress”-RPE1, AGS (gray) and “intrinsic high-replication stress”- GSU, KE39 (green). Data expressed as mean fluorescence intensity of pKAP1 signal per cell ±S.D from the following number of cells- RPE1: Control-718, 0.2nM-888, 2.0nM-524, 20.0nM-570, 200.0nM- 350; AGS: Control-2080, 0.2nM-1487, 2.0nM-1996, 20.0nM-1211, 200.0nM- 1396; GSU: Control-2092, 0.2nM-3319, 2.0nM-4035, 20.0nM-2057, 200.0nM-2402; KE39: Control-577, 0.2nM-1004, 2.0nM-1022, 20.0nM-728, 200.0nM-958. (H) Representative immunofluorescence images of pH2AX (red) and pKAP1 (green) stainings in prexasertib (20nM) treated cell lines with intrinsic “low-replication stress”-RPE1, AGS, and intrinsic “high-replication stress”- GSU, KE39; Scale bar: 50μm.

Article Snippet: ; p value calculated by unpaired t test. (I) MK-1775 (Wee1) sensitivity between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.

Techniques: Expressing, Activity Assay, Immunofluorescence, Staining, Fluorescence, Control

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Gastric cancer with ploidy abnormalities and high H2AX expression are more sensitive to DDR pathway inhibitors (A) Correlation plot between aneuploidy score and PF-477736 (Chk1/2) sensitivity (log2 fold change) for the CCLE gastric cancer cell lines; cell lines used in this study marked in red; R squared value and p value calculated by simple linear correlation analysis. (B) Prexasertib (Chk1/2) sensitivity between “ TP53 wt” and “ TP53 mut” groups of gastric cancer cell lines BROAD institute PRISM repurposing drug screen dataset; difference between the prexasertib (Chk1/2) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (C) PF-477736 (Chk1/2) sensitivity between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the PF-477736 (Chk1/2) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (D) PF-477736 (Chk1/2) sensitivity between low aneuploidy score (gray) and high aneuploidy score (red) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the PF-477736 (Chk1/2) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (E) PF-477736 (Chk1/2) sensitivity between “low H2AX expressing” (gray) and “high H2AX expressing” (green) groups (log2(TPM+1)) of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the PF-477736 (Chk1/2) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (F) Dose-response curve of non-neoplastic cell line RPE1, low replication-stress (gray)-AGS, and high replication-stress (blue)-KE39, GSU gastric cancer cell lines to indicated concentrations of prexasertib (Chk1/2); best-fit IC50 scores are displayed; data presented as mean ± S.D. of four culture replicates at each indicated dose. (G) Correlation plot between aneuploidy score and MK-1775 (Wee1) sensitivity (log2 fold change) for the CCLE gastric cancer cell lines; cell lines used in this study marked in red; R squared value and p value calculated by simple linear correlation analysis. (H) MK-1775 (Wee1) sensitivity between “ TP53 wt” and “ TP53 mut” groups of gastric cancer cell lines BROAD institute PRISM repurposing drug screen dataset; the difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (I) MK-1775 (Wee1) sensitivity between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (J) MK-1775 (Wee1) sensitivity between low aneuploidy score (gray) and high aneuploidy score (red) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. K) MK-1775 (Wee1) sensitivity between “low H2AX expressing” (gray) and “high H2AX expressing” (blue) groups (log2(TPM+1)) of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.; p value calculated by unpaired t test. (L) Dose-response curve of non-neoplastic cell line RPE1, low replication-stress (gray)-AGS, and high replication-stress (blue)-KE39, GSU gastric cancer cell lines to indicated concentrations of MK-1775 (Wee1); best-fit IC50 scores are displayed; data presented as mean ± S.D. of four culture replicates at each indicated dose.

Article Snippet: ; p value calculated by unpaired t test. (I) MK-1775 (Wee1) sensitivity between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.

Techniques: Expressing

Journal: iScience

Article Title: Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

doi: 10.1016/j.isci.2023.108169

Figure Lengend Snippet: Combination of DDR pathway inhibitors and irinotecan improves anti-tumor activity in gastric cancer models compared to monotherapy (A) Heatmap table representing the correlation between the Cumulative score of ploidy abnormalities and prexasertib sensitivity for gastric cancer cell lines in CCLE. The scoring scheme is as follows: TP53 status: wt = 1, mut = 2; Aneuploidy score: 15 and less than 15 = 1, more than 15 = 2; High amplitude focal amplifications: 20 and less than 20 = 1, more than 20 = 2; Ploidy score: 2.5 and less than 2.5 = 1, more than 2.5 = 2; WGD: No WGD = 0, WGD 1 = 1, WGD2 = 2; CIN25 score: 6.1 and less than 6.1 = 1, more than 6.1 = 2; H2AX Expression: 6 and less than 6 = 1, more than 6 = 2. R squared value and p value calculated by simple linear correlation analysis. (B) Prexasertib sensitivity between three sub-ranged “Cumulative score of ploidy abnormalities” groups of gastric cancer cell lines BROAD institute PRISM repurposing drug screen dataset; difference between the prexasertib (Chk1/2) sensitivity (log2 fold change) is represented as the mean ± S.D.; L = low, M = medium, H = high; p value calculated by unpaired t test. (C) KE39 cells were treated with increasing concentrations of prexasertib (left) or MK1775 (right) in combination with increasing concentrations of SN38. Viability in the treatment groups was normalized to DMSO control. Analysis of inhibition-synergistic effect was performed by SynergyFinder using Zero Interaction Potency (ZIP) model. The inhibition rate was used to calculate ZIP synergy score. The white box indicates area (doses) with most drug synergy. (D) Cell viability of patient-derived gastric cancer organoids treated with vehicle, SN38, prexasetib, MK1775, prexasetib+SN38 and MK1775+SN38. Dark organoids indicate apoptotic organoids. Data presented as mean ± S.D; p value calculated by unpaired t test. Representative images below. Scale bar: 50 μm. (E) A schematic showing the timeline of the KE39 xenograft mouse experiment (n = 6 per group). (F) Change in xenograft volumes in mice treated with indicated control or drug(s) over time. Data are mean ± SEM; p value calculated by two-way ANOVA test. (G) Measurement of tumor weights at the end of the treatment in indicated control and treatment groups. Data are mean ± SEM; p value calculated by Mann-Whitney test. (H) Images of dissected tumors from mice treated with indicated control or treatment group.

Article Snippet: ; p value calculated by unpaired t test. (I) MK-1775 (Wee1) sensitivity between “No WGD (0)” (gray) and “with WGD (1/2)” (green) groups of gastric cancer cell lines available in BROAD institute PRISM repurposing drug screen dataset; difference between the MK-1775 (Wee1) sensitivity (log2 fold change) is represented as the mean ± S.D.

Techniques: Activity Assay, Expressing, Control, Inhibition, Derivative Assay, MANN-WHITNEY

Journal: Oncotarget

Article Title: Distinct mechanisms contribute to acquired cisplatin resistance of urothelial carcinoma cells

doi: 10.18632/oncotarget.9321

Figure Lengend Snippet: ( A – D ) J-82 cells and CisPt resistant cells (J-82 R ) were treated with different concentrations of the pan Chk inhibitor AZD-7762 (A), the Chk1-specific inhibitor LY2603618 (B), the Wee1 kinase inhibitor MK-1775 (C) or the cyclin dependent kinase inhibitor roscovitine (D). After an incubation period of 72 h in the presence of the corresponding inhibitor, viability was analyzed using the Alamar blue assay. Data shown are the mean ± SD from three independent experiments, each performed in quadruplicate. *statistical significance of parental cells vs. CisPt resistant cells. *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05. ( E – F ) J-82 parental and CisPt resistant cells (J-82 R ) were pre-treated with 0.4 μM of the pan Chk inhibitor AZD-7762 (E) or the Chk1-specific inhibitor LY2603618 (F) for 1 h followed by the addition of CisPt (2 μM). After further incubation period of 72 h in the absence of the Chk inhibitor, cell viability was analyzed using the Alamar blue assay. Relative viability in the corresponding inhibitor-only treated controls was set to 100%. Data shown are the mean ± SD from three independent experiments, each performed in quadruplicate. *statistical significance of parental cells vs. CisPt resistant cells. ** p ≤ 0.01; * p ≤ 0.05.

Article Snippet: ATM/ATR inhibitor VE-822 (CatNo:S7102) and Wee1 kinase inhibitor MK-1775 (CatNo: S1525) were obtained from Selleckchem (Munich, Germany), lovastatin (CatNo: M2147), cyclin-dependent kinase inhibitor roscovitine (CatNo: R7772) and the pan (i.e. Chk1 and Chk2) checkpoint kinase (Chk) inhibitor AZD-7762 (CatNo: SMLO350) from Sigma Aldrich Life Science (Darmstadt, Germany), Rad51 inhibitor RI-1 (CatNo: 553514) from Calbiochem (San Diego, CA, United States), Chk1-specific inhibitor LY2603618 (CatNo: A8638) and PARP-1 inhibitor olaparib (CatNo: A4154) are from Apexbio (Houston, TX, USA) and the Raf kinase inhibitor sorafenib was obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany) (CatNo: Sc-220125).

Techniques: Incubation, Alamar Blue Assay

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Sequencing

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Association of WEE1 expression with the clinicopathological features and prognosis of CRC. ( a ) Immunohistochemical staining of WEE1 in surgical CRC specimens (100× magnification). Based on the staining intensity, the cells were classified as strong, moderate, weak, and negative, with strong and moderate being classified as the WEE1-positive group and weak and negative as the WEE1-negative group. ( b ) Immunohistochemical staining of p53 in surgical CRC specimens (100× magnification). Based on the distribution of immunostained positive cells, the cells were classified as diffuse strong, nested, negative, mosaic, sporadic, and diffuse weak, with diffuse strong, nested, and negative being classified as the p53 mutations and mosaic, sporadic, and diffuse weak as p53 wild types. ( c ) Database analysis based on the UCSC cancer genomic browser relationship between WEE1 expression and prognosis (overall survival rate) in CRC (GDC, The Cancer Genome Atlas (TCGA), Colon Adenocarcinoma (COAD) n = 487). ( d ) Association between WEE1 expression and prognosis (overall survival rate) in 152 patients with surgically resected CRC at our hospital. ( e ) Comparison of the overall survival rates of patients ( n = 152) who underwent surgery for CRC classified by WEE1 expression and KRAS mutation status. Log-rank test *: p < 0.05, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, Comparison, Mutagenesis

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: WEE1 expression and clinicopathological features.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: WEE1 expression and gene mutation status.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Mutagenesis

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Quantification of WEE1 expression in CRC cell lines and effect of WEE1 inhibitor on cell proliferation. ( a ) Relative expression levels of WEE1 in various CRC cell lines measured using real-time PCR, where its expression level in normal colonic mucosa was set as 1. ( b ) Growth curves of various CRC cell lines after exposure to various concentrations of the WEE1 inhibitor, adavosertib (200, 500, and 1000 nM), and untreated cells (control). The mean values are plotted. One-way ANOVA followed by Dunnett’s post hoc test, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Effects of the WEE1 inhibitor on cell cycle and apoptosis induction in CRC cell lines. ( a ) Changes over time in the percentage of cells in the G1, S, and G2/M phases of the cell cycle as measured by cell cycle analysis in untreated (0 h) and various adavosertib-treated (1000 nM) CRC cell lines (at 4, 12, and 24 h). Comparison of percentage of apoptotic cells before (0 h) and 24 h after addition of adavosertib. *: p < 0.05, n.s.: not significant. ( b ) Changes over time in the percentage of apoptotic, viable, and necrotic cells in various untreated (0 h) and adavosertib-treated colon cancer cell lines. The cell lines were treated with 1000 nM adavosertib, and the analysis was performed 6, 24, and 48 h after treatment. Comparison of percentage of apoptotic cells before (0 h) and 48 h after the addition of adavosertib. *: p < 0.05, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Cell Cycle Assay, Comparison

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Effects of the WEE1 inhibitor, adavosertib, on tumor volume in an orthotopic transplanted mouse model of CRC. ( a ) Orthotopic transplanted mouse model of CRC using MC38: transplant design and treatment schedule. n = 5, control group; n = 4, treatment group. ( b ) Macroscopic images of mouse tumors obtained following euthanasia on day 35 post-transplantation. ( c ) Comparison of tumor volume between control and treatment groups. ( d ) Orthotopic transplanted mouse model of CRC using CT26: transplant design and treatment schedule. n = 6, control group; n = 6, treatment group. ( e ) Macroscopic images of mouse tumors obtained following euthanasia on day 24 post-transplantation. ( f ) Comparison of tumor volume between control and treatment groups. Welch’s t -test, **: p < 0.01, n.s.: not significant.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control, Transplantation Assay, Comparison

Journal: Cancers

Article Title: WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations

doi: 10.3390/cancers16183136

Figure Lengend Snippet: Evaluation of the gene expression profiles in transplanted tumors using RNA sequencing. ( a ) Statistics of differentially expressed genes (DEGs; p < 0.05 and fold change > 2). ( b ) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. ( c ) Gene Ontology (GO) enrichment analysis. ( d ) Gene Set Enrichment Analysis (GSEA) in orthotopically transplanted tumors treated with or without WEE1 inhibitor. NES: normalized enrichment score.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775) was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Gene Expression, RNA Sequencing

Journal: Molecular cancer therapeutics

Article Title: Functional genetic screen identifies increased sensitivity to WEE1 inhibition in cells with defects in Fanconi Anaemia and HR pathways

doi: 10.1158/1535-7163.MCT-14-0845

Figure Lengend Snippet: Drug effect Z-scores for an siRNA/MK-1775 WEE1 inhibitor sensitivity screen in WiDr cells. Z-scores <−2 represent statistically significant sensitising effects to 200 nM MK-1775 (dotted line). Black dots, individual siRNA SMARTpools targeting 1206 genes; red dots, hits selected for further validation.

Article Snippet: Chemical inhibitors and antibodies The following chemical inhibitors were used at the indicated concentrations, unless stated otherwise, WEE1 inhibitor MK-1775 (250nM, Axon Medchem), CDK1 inhibitor RO-3306 (10μM; Tocris), hydroxyurea (HU; 3mM), mitomycin C (MMC, 100nM; both from Sigma-Aldrich).

Techniques: Biomarker Discovery

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