Journal: bioRxiv

Article Title: MYC amplifies mitotic perturbations elicited by LXY18 to enable synthetic lethality

doi: 10.1101/2023.11.07.565938

Figure Lengend Snippet: RPE-NEO and RPE-MYC cells were treated with either vehicle control (0.1% DMSO) or indicated mitotic inhibitors for 24 h before immunofluorescent staining for H3Ser10P and staining for DNA with 4’,6-diamidino-2-phenylindole (DAPI). Cells positive for H3Ser10P were scored as mitotic cells and quantified when cells were treated with compounds other than AURKB inhibitors. For cells exposed to AZD1152 and AMG900, mitotic cells were identified based on their condensed chromosomes revealed by DAPI staining. The following mitotic inhibitors were used at minimally effective concentrations that are known to inhibit their respective targets, AZD1152 (200 nM), AMG900 (10 nM), BAY1217389 (20 nM), Centrinone (1 μM), GSK923295 (40 nM), SB743921 HCl (1.25 nM), Sovilnesib (80 nM). (A) Representative immunofluorescent images. (B) The percentage of the mitotic cells. Data are presented as mean ± SD. ****p<0.0001. (C) Histograms show the ratio of the mitotic index (MI) in RPE-MYC cells relative to RPE- NEO cells.

Article Snippet: No. T14927), GSK923295 (TargetMol, CAS: 1088965-37-0, Cat. No. T2039), SB743921 HCl (TargetMol, CAS: 940929-33-9, Cat. No. T2255), and Sovilnesib (TargetMol, CAS: 2410796-79-9, Cat. No. T39994).

Techniques: Staining

Journal: Nature Communications

Article Title: CRISPR knockout screening identifies combinatorial drug targets in pancreatic cancer and models cellular drug response

doi: 10.1038/s41467-018-06676-2

Figure Lengend Snippet: The validation of CRISPR screening hits. a , b Kaplan–Meier plots showing the survival analysis of TCGA data for pancreatic cancer patients expressing high levels of CENPE and RRM1 genes. High expression: >1 standard deviation (SD), and higher expression: >2 SD above the population mean. The number of patients in each group is indicated with n . c Box plots show fold change distribution of sgRNAs targeting CENPE ( n = 10), RRM1 ( n = 10), and control sgRNAs ( n = 100) in tumors from control-treated and trametinib-treated mice. Bounds of the box spans from 25 to 75% percentile, center line represents median, and whiskers visualize 5 and 95% of the data points. Significance was assessed by Kolmogorov–Smirnov test. d MTT assay-based relative viability/growth inhibition levels are shown in PDX366 and mPanc96 cells expressing WT Cas9 with control sgRNA or two separate sgRNAs targeting CENPE (top) and RRM1 genes (bottom). e Relative viability/growth inhibition levels are shown for three different cell lines, PDX366, MPANC-96, and BxPC3 cells, treated with control, MEK inhibitor (trametinib), CENPE inhibitor (GSK923295), and combined MEK inhibitor and CENPE inhibitor. f RRM1 inhibitor (COH29) and trametinib were tested together in PDX366 and MPANC-96 cells. g Bar plots show relative growth inhibition of MPANC-96 cells treated with inhibitors of Aurora A/B (ZM447439) and PLK1 inhibitor (BI-2536) alone or in combination with trametinib. Each experiment was repeated at least three times and error bars represent the standard error of the mean value. CI cytotoxicity index, Comb combination of two drugs

Article Snippet: Trametinib (EuroAsia TransContinental, Mumbai, India), GSK923295 (ChemieTek, Indianapolis, IN, USA), Aurora A/B inhibitor (ZM447439, Selleckchem, cat. no. S1103), and PLK1 inhibitor (BI-2536, MedChemExpress, cat. no. HY-50698) were dissolved in sterile DMSO to make 5 mM stock solutions.

Techniques: CRISPR, Expressing, Standard Deviation, MTT Assay, Inhibition

Journal: Nature Communications

Article Title: CRISPR knockout screening identifies combinatorial drug targets in pancreatic cancer and models cellular drug response

doi: 10.1038/s41467-018-06676-2

Figure Lengend Snippet: Combinatorial MEK and CENPE inhibition results in synergistic cell death. a Each frame represents movie stills from time-lapse longer-term live-cell imaging as cells undergo mitosis starting from nuclear envelope break down (NEBD) to exit from mitosis. b Each bar graph show total time duration of each of the individually tracked cells spent in mitosis. The NEBD was taken as the beginning of mitosis. Individual cells ( n = ~20/treatment group) were manually tracked from lime-lapse movies until they exit mitosis (anaphase) or died in mitosis for each of the treatment groups. c Flow cytometry profiles of DNA content in PDX366 PDAC cells treated with control, single-agent, or combinatorial MEK and CENPE inhibitors are shown. d Bar graphs represent treatment-mediated percent changes in cells with 2n (G1), 4n (G2), and >4n (polyploidy) DNA. e Immunofluorescent images of PDX366 PDAC cells treated with control and combinatorial (trametinib and CENPE inhibitors) are shown after DAPI and tubulin staining in the first two left columns respectively. f Representative hematoxylin and eosin (H&E)-stained tumor sections are shown. Tumors were harvested from mice that had undergone 72 h of treatment with 125 mg/kg CENPE inhibitor alone or in combination with 0.3 mg/kg trametinib. Arrows indicate mitotic figures. g Dot-plot showing the number of mitotic cells quantified from 10 different high-power imaging fields (HPFs) per tumor section. h Results show MRI-measured effects of single and combination of drugs on tumor formation. At 1 week after orthotopic implant, mice received control, MEK inhibitor (trametinib), CENPE inhibitor (GSK923295), or combined MEK and CENPE inhibitor. Results show MRI-measured tumor volumes after 4 weeks of treatment. i MRI-measured tumor volumes are shown in mice where tumors were allowed forming for 4 weeks and the treatments (as in e ) were started. Beginning of treatment is marked with an arrow. Bounds of the box spans from 25 to 75% percentile, center line represents median, and whiskers visualize 5 and 95% of the data points. Individual data points are marked with circles or dots. Error bars represent the standard error of the mean value of at least three independent experiments. Symbols * and *** denote P < 0.05 and P < 0.001, respectively

Article Snippet: Trametinib (EuroAsia TransContinental, Mumbai, India), GSK923295 (ChemieTek, Indianapolis, IN, USA), Aurora A/B inhibitor (ZM447439, Selleckchem, cat. no. S1103), and PLK1 inhibitor (BI-2536, MedChemExpress, cat. no. HY-50698) were dissolved in sterile DMSO to make 5 mM stock solutions.

Techniques: Inhibition, Live Cell Imaging, Flow Cytometry, Staining, Imaging