Journal: The EMBO Journal

Article Title: Structural insights into the activation mechanism of antimicrobial GBP1

doi: 10.1038/s44318-023-00023-y

Figure Lengend Snippet: Reagents and tools.

Article Snippet: Guanosine diphosphate (GDP) , Jena Bioscience , NU-1172.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Structural and functional insights into Pseudomonas aeruginosa ApaH , a diadenosine tetraphosphatase crucial for bacterial virulence

doi: 10.1002/pro.70781

Figure Lengend Snippet: Kinetic analysis and substrate specificity of Pseudomonas aeruginosa diadenosine tetraphosphatase ( Pa ApaH). (a) Hydrolysis of diadenosine tetraphosphate (Ap4A) by Pa ApaH followed by ion‐pair reversed‐phase high‐performance liquid chromatography. Chromatograms of the reaction mixture (5 nM Pa ApaH in 20 mM Tris–HCl pH 7.4, 100 mM NaCl, 2 mM MnCl 2 and 20 μM Ap4A) before (dark blue) or after (light blue) 10 min‐incubation at 20°C. The gray dashed line shows elution of the ADP standard (20 μM). (b) Michaelis–Menten plot of Pa ApaH (5 nM) hydrolase activity measured with Ap4A as substrate in the same condition as panel (a). Initial velocities were determined at varying Ap4A concentrations (mean ± standard deviation [SD]; n = 3). (c) Substrate specificity of Pa ApaH. Histograms represent substrates concentration before (dark color) and after (light color) 10 min‐incubation with Pa ApaH in the same conditions as panel (a) (mean ± SD; n = 3). Unpaired Student's t ‐test was used to assess statistical significance (* p < 0.05; **** p < 0.001). (d–f) Chromatograms of the reaction mixture before (dark spectra) or after (light spectra) 10 min‐incubation at 20°C with Ap3A, Ap5A, or Gp4G in place of Ap4A, respectively. Reactions were carried out in the same conditions as panel (a). The gray dashed line shows elution of the nucleotide standards.

Article Snippet: Final concentrations were: Pa ApaH 5 nM; Ap4A (Jena Bioscience, Jena, Germany) 1, 2, 4, 5, 10, 20, 40, and 80 μM.

Techniques: High Performance Liquid Chromatography, Incubation, Activity Assay, Standard Deviation, Concentration Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Structural and functional insights into Pseudomonas aeruginosa ApaH , a diadenosine tetraphosphatase crucial for bacterial virulence

doi: 10.1002/pro.70781

Figure Lengend Snippet: Docking analysis of diadenosine tetraphosphate (Ap4A) and other substrates. (a) Computationally predicted Ap4A binding poses to subsite 1 and subsite 2. The conformation obtained by classical docking prediction ( OUT —light gray sticks) is superposed with the one obtained by induced fit docking (IFD) ( IN —blue sticks). The protein is shown in dark green surface representation to better highlight the substrate binding groove. (b) Ap4A docking pose obtained by IFD. The side chains of the residues that shape the binding cleft and predicted to interact with Ap4A are shown as stick. The adenine base binding in subsite 1 is labeled A1, whereas the one binding in subsite 2 is labeled A2. H‐bonds interactions are shown as green dashed lines. The five residues that were selected for mutagenesis analysis (see below) are highlighted in orange. (c) Superposition of the proposed IFD conformations for Ap4A (blue), Ap3A (purple) and Ap5A (orange). (d) Superposition of the proposed IFD conformations for Ap4A (blue) and Gp4G (green).

Article Snippet: Final concentrations were: Pa ApaH 5 nM; Ap4A (Jena Bioscience, Jena, Germany) 1, 2, 4, 5, 10, 20, 40, and 80 μM.

Techniques: Binding Assay, Labeling, Mutagenesis

Journal: Protein Science : A Publication of the Protein Society

Article Title: Structural and functional insights into Pseudomonas aeruginosa ApaH , a diadenosine tetraphosphatase crucial for bacterial virulence

doi: 10.1002/pro.70781

Figure Lengend Snippet: In vivo and in vitro characterization of Pseudomonas aeruginosa diadenosine tetraphosphatase ( Pa ApaH) mutant variants. (a) Pyoverdine levels, (b) pyocyanin levels, and (c) swarming motility of Pa PAO1 wild‐type (WT) and the isogenic mutant Δ apaH carrying the empty plasmid pME6032 (pME) or its derivatives pME apaH , pME apaH R41A , pME apaH R183A , pME apaH R185A , pME apaH K198A , and pME apaH D245A as indicated. Values are the mean (± standard deviation [SD]) of three independent experiments. Asterisks indicate a statistically significant difference compared to Δ apaH carrying pME apaH ( ***p < 0.001; analysis of variance). Images in panel (c) are representative of three independent experiments giving similar results. (d) Hydrolysis of diadenosine tetraphosphate (Ap4A) by Pa ApaH mutants: Percentage activity with respect to the WT measured for the five variants by ion‐pair reversed‐phase high‐performance liquid chromatography in the same conditions as Figure . Values are the mean (± SD) of three experiments. Statistical significance was determined using unpaired Student's t ‐test (**** p < 0.0001). Throughout the figure the results of the two variants R185A and D245A are highlighted in red. OD 405 , optical density at 405 nm; OD 600 , optical density at 600 nm.

Article Snippet: Final concentrations were: Pa ApaH 5 nM; Ap4A (Jena Bioscience, Jena, Germany) 1, 2, 4, 5, 10, 20, 40, and 80 μM.

Techniques: In Vivo, In Vitro, Mutagenesis, Plasmid Preparation, Standard Deviation, Activity Assay, High Performance Liquid Chromatography

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Western blot of shControl, shMAP3K7, shCHD1, and shMAP3K7-shCHD1 (shDouble) LNCaP cells. Multiple shRNA constructs are shown. (B) 5-day growth assay for LNCaP shControl, shMAP3K7, shCHD1, and various shDouble combinations in steroid-depleted medium with vehicle (0.1% EtOH) or 10 pM R1881. Asterick denotes shDouble (2) combination used in subsequent experiments. (n=3; p-value indicated for all shDouble constructs versus shControl at Day 5). (C) Quantification of day 5 from growth assay in B, including the shDouble-2 combination (n=3; p-values indicate comparison to shControl EtOH). (D) Day 7 of R1881 dose response curves for LNCaP cells normalized to respective EtOH treatment (n=3; p-values indicate comparison to shControl at each dose). (E) RT-qPCR of indicated genes in LNCaP cells cultured in steroid-depleted medium ± EtOH or 1 nM R1881 for 8 hr. Ct values normalized to B2M then to shControl (n=3; p-values represent comparison to shControl EtOH). (F) Quantification (n=3) and representative western blot of AR protein in shControl and shDouble LNCaP cell lysates isolated after different treatments: complete growth medium or steroid-depleted medium ± 1 nM R1881 for 4 hr or 24 hr. (G) Quantification (n=3) and representative western blot of AR protein in nuclear fractions isolated from LNCaP cell lysates after treatment with ± 1 nM R1881 for 4 hr. AR blot of EtOH-treated samples is shown at a higher exposure than R1881-treated samples in order to visualize bands. All data represent mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test (C, G), two-way ANOVA with Dunnett’s multiple comparisons test (B, D, E), and unpaired two-tailed t-test (F); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: Western Blot, shRNA, Construct, Growth Assay, Quantitative RT-PCR, Cell Culture, Isolation, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Day 5 of growth assay for indicated LNCaP cells treated with 5-60 µM enzalutamide in complete medium, normalized to DMSO vehicle control (n=3; p-values indicate comparison to shControl at each concentration). (B) RT-qPCR of AR-FL and AR-v7 in indicated LNCaP cells ± 1 nM R1881 for 4 h. (C) RT-qPCR analysis of AR-v7 target genes UGT2B17, AKT1, and UBE2C in shControl and shDouble LNCaP cells ± EtOH or 1 nM R1881 for 4 h. For (B-C), Ct values normalized to B2M expression and then to shControl (n=3; p-values indicate comparison to shControl EtOH). (D) Correlation of UGT2B17, (E) AKT1, and (F) UBE2C mRNA expression with quartiles of MAP3K7 and CHD1 mRNA co-expression in the MSKCC dataset (25). (G) Propidium iodide (PI) counts normalized to cell confluence of shControl (left) and shDouble (right) LNCaP cells in castration conditions beginning 24 hours after transfection with indicated siRNAs (n=3; p-values indicate comparison to siCtrl). (H) Western blot of lysates from LNCaP cells 96 hours after transfection with indicated siRNAs showing cleaved PARP, total PARP, and the loading control β-actin. All data represent means ± SEM; unpaired two-tailed t-tests (D, E, F) and two-way ANOVA with Dunnett’s multiple comparisons test (A, B, C, G); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: Growth Assay, Concentration Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Schematic of experimental setup and workflow for RNA-Seq and ChIP-seq. (B) Heatmap illustration of RNA-Seq z-score mRNA expression changes of a validated AR target gene signature in indicated cell types ± 1 nM R1881 for 4 hr (33). (C) GSEA of AR signaling gene signature (MSigDB: M15861) in indicated TCGA PCa groups with copy number loss of MAP3K7, CHD1, or both versus PCa with loss of neither. (D) Normalized signal intensity (read count per million mapped reads) of AR ChIP-seq binding events centered on the peak regions in indicated cells treated with vehicle EtOH (top) or 1 nM R1881 (bottom). (E) Venn diagram of differential, gene-annotated AR ChIP binding sites in indicated cell types treated with 1 nM R1881 compared to shControl EtOH as a background (fold-change >4, p-value <1e-04). (F) Venn diagram of the annotated AR ChIP sites from (E) that are further filtered for significant gene expression changes (FDR<0.05) over shControl EtOH. (G) Scatter plots of annotated genes from (F) including the shControl R1881 versus shControl EtOH comparison not shown in (F). Genes with log2 fold-change ± 1.5 are shown in green.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Binding Assay

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Pathway enrichment analysis for LNCaP shMAP3K7 versus shControl (+ R1881) RNA-Seq changes using Metascape (28). (B) Summary of GSEA results comparing TCGA cases with lower quartile mRNA expression of MAP3K7, CHD1 or both versus cases with upper quartile expression of respective groups. Nominal p-value <0.05 for all enrichments included. (C) GSEA plots of signatures particularly enriched in TCGA cases with low versus upper quartile expression of MAP3K7. (D) GSEA plots of the G2M signature in TCGA MAP3K7 low versus high cases and LNCaP shMAP3K7 versus shControl cells (+R1881). (E) Flow cytometry cell cycle analysis of indicated LNCaP cells after 48 hours in steroid-depleted medium ± 1 nM R1881 showing percent of total cells in G2M (n=3). Data represent mean ± SEM. Log-rank Mantel-Cox test (A, B), One-way ANOVA with Tukey’s multiple comparisons test (G); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: RNA Sequencing Assay, Expressing, Flow Cytometry, Cell Cycle Assay