Journal: bioRxiv

Article Title: A drug-screening platform based on organotypic cultures identifies vulnerabilities to prevent local relapse and treat established brain metastasis

doi: 10.1101/2020.10.16.329243

Figure Lengend Snippet: (A) Schema of experimental design. (B) Quantification of bioluminescence emitted by H2030-BrM established metastases in brain organotypic cultures. BLI values were obtained 6h after the addition of DEBIO-0932 or DMSO normalized by the values of each culture before any treatment. Data is shown as relative to DMSO BLI values in box-and-whisker plots where every dot represents an organotypic culture and the line in the box corresponds to the median. Whiskers go from the minimum to the maximum value (n=20 organotypic cultures per experimental condition, 3 independent experiments). P value was calculated using two-tailed t-test. (C) Representative image of a fully established brain metastasis from H2030-BrM before and after laser capture microdissection (LCM). The dotted line delimits the metastasis. Scale bar, 100 μm. (D) Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log 2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown. (E) Representative images showing RPLP1, AHR, UBE4B and DDA1 levels in brain metastases (generated by intracardiac inoculation of H2030-BrM) found at endpoint of vehicle and DEBIO-0932 treated animals. This result was reproduced in 2 independent staining with different brains. BB: bisbenzamide. Scale bars, 50 μm; high magnification, 12 μm. (F) Quantification of percentage of nuclear DDA1+ BB+ cells shown in (E). Values are shown in box-and-whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. Whiskers go from the minimum to the maximum value (n=16 metastatic lesions from 4 brains per condition, 2 independent staining with different brains were performed). P value was calculated using two-tailed t-test. (G) Quantification of RPLP1 and AHR levels shown in (E) in arbitrary fluorescent units (A.F.U.). Values are shown in box- and-whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. Whiskers go from the minimum to the maximum value (n=8-16 metastatic lesions from 2-4 brains per condition, 2 independent staining with different brains were performed). P value was calculated using two-tailed t-test. (H) GSEA of top 25 upregulated (red) and downregulated (green; only four fulfill the filter) pathways upon DEBIO-0932 treatment. Those biological processes represented with more than one signature are labelled with colored lines. (I) Examples of signatures included in the main biological processes represented in the proteomic analysis. GO:0000981 corresponds to the Gene Ontology signature “DNA binding, Transcription factor activity, RNA polymerase II specific”. (J) Representative images showing p-ERK levels in organotypic cultures from (B). This result was reproduced in 3 independent staining with organotypic cultures from different mice. Scale bar, 20 μm. (K) Immunohistochemistry against UBE4B and AHR in 16 human brain metastases with (green) and without (red) HSP90-dependent oncogenic drivers. No molecular information available for the sample depicted in gray. Scale bar, 50 μm. (L) Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis (SPBM) and high expression levels of UBE4B (upper panel) or DDA1 (lower panel) in a cohort of 21 breast cancer brain metastasis patients.

Article Snippet: Primary antibodies: GFP (1:1,000; GFP-1020, Aves Labs), BrdU (1:500; ab6326, Abcam), Ki67 (1:500; ab15580, Abcam), HSP90α/β F-8 (1:500; sc-13119; Santa Cruz Biotechnology), HSP70/ HSC70 W27 (1:500; sc-24; Santa Cruz Biotechnology), AHR (1:300; 31.714.200, US Biological), UBE4B (1:100; ab97697; Abcam), RPLP1 (1:100; HPA003368, Sigma-Aldrich), DDA1 (1:100; 14995-1-AP; ProteinTech), NeuN (1:500; MAB377, Millipore), collagen IV (1:1000; AB756P, Millipore).

Techniques: Whisker Assay, Two Tailed Test, Laser Capture Microdissection, Generated, Staining, Binding Assay, Activity Assay, Immunohistochemistry, Expressing

Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Evolutionary analyses of divergent mammalian HBV X proteins. (A) Phylogenetic analysis of the X proteins from hepadnaviruses that naturally infect mammals. The viral X proteins tested in our in vitro functional assays (Fig. 5 to ​to7)7) are indicated by an asterisk. Phylogenetic analysis of orthohepadnaviral X proteins was performed using a 161-amino-acid alignment obtained with MUSCLE (see supplemental data set 2 at https://figshare.com/articles/DatasetS2_Orthohepadnaviral_HBx_amino_acid_alignment_interleaved_phylip_format_/6194825) and the tree was built with PhyML and a JTT+I+G model with 1,000 bootstrap replicates. Bootstrap values (>800/1,000) are indicated at the nodes. The tree was rooted for representation purposes according to the work of Drexler et al. (52) (but the outgroup of orthohepadnavirus is still under debate [2]). The scale bar indicates the number of amino acid substitutions per site. We analyzed the X proteins from HBVs from the ground squirrel (GSHBV), arctic squirrel (ASHBV), and woodchuck (WHV), three bat viruses naturally infecting Hipposideros cf. ruber (roundleaf bat), Rhinolophus alcyone (horseshoe bat), and Uroderma bilobatum (tent-making bat), respectively (RBHBV, HBHBV, and TBHBV), wooly monkey HBV (WMHBV), human HBV, and HBVs from other indicated hominoids. (B) Amino acid alignment of the viral X proteins used for Fig. 5 to ​to7.7. The black-to-white gradient depicts high-to-low sequence identity (Geneious). The open reading frames (ORFs) overlapping with HBx are shown, as well as the DDB1-binding region in the human viral HBx protein (72).

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: In Vitro, Functional Assay, Sequencing, Binding Assay

Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Highly divergent mammalian HBV X proteins show a conserved property of recruiting human DDB1 and antagonizing human Smc5/6 restriction. (A and B) Degradation of the human Smc5/6 complex by mammalian hepadnavirus X proteins. Human hepatoma HepG2 cells (A) and 293T cells (B) were transduced with a lentivector expressing only GFP (control) or the GFP-fused X protein from diverse hepadnaviruses (Fig. 4) or a mock control. Western blot analysis of the endogenous Smc6 and Nsmce4A was performed (see Materials and Methods). GAPDH served as a loading control. (C) Effect of mammalian X proteins on transiently transfected reporter gene activity. HepG2 cells were transfected with a luciferase reporter construct and the next day transduced with lentiviral vectors expressing the indicated proteins as described above. At days 5 to 7, the luciferase activity was measured; the fold increase of relative light units (RLU) versus the GFP control condition (set at 1) is shown. The means from three independent experiments are shown, along with SDs. *, P value = 0.1. P values correspond to the Wilcoxon Mann-Whitney test against the null hypothesis of no difference in the luciferase activity between the GFP control and GFP-X conditions. Of note, the same six X proteins unfused to GFP (i.e., in their native forms) also retained this activity (data not shown). (D) Interaction with human DDB1 protein was conserved for all hepadnaviral X proteins tested. The presence of DDB1 and GFP-fused protein (IP) was assessed by Western blotting. The viral X proteins could all interact with human DDB1, except for the DDB1 binding-deficient HBx mutant (R96E) that was used as a control. Note that GFP migrates to a position near the immunoglobulin light chain.

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: Transduction, Expressing, Western Blot, Transfection, Activity Assay, Luciferase, Construct, MANN-WHITNEY, Binding Assay, Mutagenesis

Journal: Frontiers in Immunology

Article Title: AMBRA1 drives gastric cancer progression through regulation of tumor plasticity

doi: 10.3389/fimmu.2024.1494364

Figure Lengend Snippet: KD of AMBRA1 triggered the FoxO3A/p53/CRK/CDKN1B signaling pathway. (A) AMBRA1 pathway network derived from the Pathway Interaction Database which was curated by STRING. (B) GSEA of transcriptomic data from gastric cancer samples of The Cancer Genome Atlas (TCGA-STAD), senescence gene sets in AMBRA1_Low vs. AMBRA1_High subgroups (n = 16 for each subgroup). (C) Correlation between AMBRA1 and FOXO3, DDB1, CRK, CDKN1B, CDK4, CCND1, BCL2L11 in TCGA-STAD on TIMER2, with Spearman’s rho value representing the correlation degree. D–I) The expression of DDB1, FOXO3, DDB1, p53, CRK, CDKN1B and Tubulin in AGS and KD-AMBRA1 cells under CHX (1 μM), MG132 (0.5 μM) and MLN4924(5 μM)-treated for 24 hours were detected by western blotting. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Rabbit antibodies, including anti-AMBRA1 (Catalog #38182), anti-p27 (Catalog #41299), anti-p38 (Catalog #33149), anti-FoxO3A (Catalog #40937), anti-CUL4A (Catalog #38477), anti-CyclinD1 (Catalog #39315), anti-DDB1 (Catalog #38485), anti-Histone H2AX (Catalog #54667), anti-Phospho-Histone H2A.X (S139) (Catalog #13343), anti-Histone H3K9me3 (Catalog #HW029), anti-FoxO3A (Catalog #40937), anti-p53 (Catalog #HW109), anti-Caspase-3 (Catalog #9662), anti-Cleaved Caspase-3 (Catalog #9661), and anti-Phospho-Thr286 CyclinD1 (Catalog #11968), were procured from Signalway Antibody LLC (SAB).

Techniques: Derivative Assay, Expressing, Western Blot