Journal: ACS pharmacology & translational science

Article Title: Stabilization of Cyclin-Dependent Kinase 4 by Methionyl-tRNA Synthetase in p16 INK4a -Negative Cancer.

doi: 10.1021/acsptsci.8b00001

Figure Lengend Snippet: Figure 5. MRS facilitates CDK4−HSP90 complex formation and stabilizes CDK4. (a) H460 cells transfected with either si-Control or si-MRS were treated with a proteasome inhibitor (MG132). Relative levels of CDK4 were quantified using ImageJ. (b) MRS, CDC37, HSP90, and CDK4 were transfected into H460 cells, and their interaction was investigated. (c) Association of MRS with CDK4, HSP90, or CDC37 was investigated via in vitro pull-down assay. (d) Effect of geldanamycin (1 μM) on CDK4 in MRS-overexpressing H460 cells. (e) Effect of MRS on the interaction between CDK4 and HSP90 measured by reconstitution of nanoluciferase luminescence. (f) H460 cells were treated with FSMO and si-MRS in combination, and their effect on the interaction between overexpressed HSP90 and CDK4 was investigated.

Article Snippet: Primary antibodies for HA (mouse, sc-7392; Santa Cruz and rabbit, H6908; Sigma), Myc (mouse, sc-40; Santa Cruz), Flag (mouse, F3165; Sigma), Strep-HRP (2-1509-001; IBA), tubulin (mouse, T6074; Sigma), β-actin (mouse, A1978; Sigma), CDK4 (mouse, sc-23896 and rabbit, sc-260; Santa Cruz), cyclin D1 (rabbit, 04-221; Merckmillipore), p-Rb (rabbit, #3590; Cell Signaling), HSP90α/β (rabbit, sc-7947; Santa Cruz), CDC37 (rabbit, sc-5617; Santa Cruz), p16 (rabbit, sc-468; Santa Cruz), CDK3 (rabbit, sc-28256; Santa Cruz), CDK6 (mouse, sc-7961; Santa Cruz), and MRS (mouse, ab50793; Abcam) and a CDK antibody sampler kit (#9868; Cell Signaling) and a Cyclin antibody sampler kit (#9869; Cell Signaling) were used to perform immunoblotting, immunoprecipitation, and immunofluorescence staining.

Techniques: Transfection, Control, In Vitro, Pull Down Assay

Journal: Molecular cancer therapeutics

Article Title: A Synergistic Anti-Cancer FAK and HDAC Inhibitor Combination Discovered by a Novel Chemical-Genetic High-Content Phenotypic Screen

doi: 10.1158/1535-7163.MCT-19-0330

Figure Lengend Snippet: A, IP of FAK from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cell lysates, immunoblotted for FAK, Cdc37, and phosphorylated Cdc37 (S13). B, IP of FAK from SCC FAK-WT cells treated with DMSO (0.1%) or 250 nM VS-4718, VS-6063, PF-562,271, or GSK2256098 for 24 h, immunoblotted for FAK, phosphorylated FAK (Y397), and Cdc37. C, Lysates from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cells, immunoblotted for FAK and phosphorylated FAK (Y397). D, IP of FAK from SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cell lysates, immunoblotted for FAK and Src. E, SCC FAK-WT cells seeded on glass coverslips, fixed, and labeled with anti-FAK (green), anti-Cdc37 (red), and DAPI (blue). Inset (right) represents dashed boxed region of main image. Arrows and arrowheads (inset) indicate examples of regions of colocalization of FAK and Cdc37. Scale bar, 20 μm. F, SCC FAK-WT, FAK−/−, FAK-G431A,F433A, and FAK-KD cells seeded on glass coverslips, fixed, and labeled with anti-FAK (green) and DAPI (blue). Scale bars, 20 μm. G, FAK synthesis and degradation profiles quantified by metabolic labeling and mass spectrometry. FAK-WT (green) and FAK-G431A,F433A (brown) synthesis and degradation curves were determined from normalized SILAC ratio profiles by nonlinear regression and plotted as means ± SEM with best fit curves and 95% confidence interval bands (n ≥ 5 peptides; representative of three independent experiments). NS, not significant (extra sum-of-squares F test). Bar chart inset summarizes inferred 50% protein turnover times for FAK-WT and FAK-G431A,F433A, plotted as means ± SD (n = 3 independent experiments). NS, not significant (two-tailed Mann–Whitney U test). H, Label-free mass spectrometric characterization of FAK-interacting proteins isolated by IP of FAK from SCC FAK-WT and FAK-G431A,F433A cell lysates. Specific FAK-binding proteins were determined versus IP from SCC FAK−/− cells (n = 3 independent experiments), satisfying Q < 0.05 (Student’s t-test with permutation-based FDR correction). Gray curves show the threshold for significant differential regulation (FDR, 5%; artificial within-groups variance, 1). Proteins satisfying P < 0.01 and fold change > 4 are labeled with gene names for clarity. I, Label-free mass spectrometric characterization of FAK-interacting proteins isolated by IP from SCC FAK-WT, FAK-KD, and FAK-G431A,F433A cell lysates and lysates from SCC FAK-WT cells treated with 250 nM VS-4718 (n = 3 independent experiments). Normalized label-free quantification of protein abundance for each protein was converted to a Z score. Differentially regulated, specific FAK-binding proteins satisfying Q < 0.05 (one-way ANOVA with permutation-based FDR correction) were hierarchically clustered and displayed as a heatmap. Proteins are labeled with gene names for clarity.

Article Snippet: Antibodies used were purchased from Cell Signaling Technology (FAK phosphorylated tyrosine-(pY)397, #3283; FAK, #3285; GAPDH, #2118; YAP, #14074; YAP phosphorylated serine-(pS)127, #13008; histone H3, #9715; histone H3 acetylated lysine-56 (K-Ac), #4243; Cdc37, #4793; Cdc37 pS13, #13248; Src, #2109; Axl #8661) or Abcam (YAP, #EP1674Y).

Techniques: Labeling, Mass Spectrometry, Two Tailed Test, MANN-WHITNEY, Isolation, Binding Assay