Journal: bioRxiv

Article Title: A viral histone-like protein exploits antagonism between linker histones and HMGB proteins to obstruct the cell cycle

doi: 10.1101/2020.12.11.418129

Figure Lengend Snippet: a. Diploid RPE-1 cell proliferation for 48 hours after treatment. The cells were transduced with recombinant adenovirus to express GFP-tagged protein VII or a GFP control. Protein VII-GFP recombinant adenovirus MOI = 50. GFP control recombinant adenovirus MOI = 50. 10 μM RO-3306 CDK-1 inhibitor treatment is a control for inhibition of proliferation and G2 arrest. At 48 hours, there was a significant difference in the total number of protein VII-GFP expressing cells compared to the no treatment control ( p <0.0001, one-way ANOVA with Tukey’s multiple comparisons test) and to the GFP control ( p <0.0001). There was no difference between the cells expressing protein VII-GFP and RO-3306 treated cells ( p =0.9933). Error bars show standard deviation. N = 4. b. Cell cycle analysis by flow cytometry of transduced RPE-1 cells shown in (a). Cells were fixed and stained with propidium iodide to assess DNA content. DNA content is plotted on the x-axis, with G1 and G2/M populations labelled, and the y-axis represents a histogram of the cell population. The y-axis is normalized to fill the entire axis independently for each sample. Since nearly 100% of transduced cells were positive for GFP expression, cells were not gated on GFP positive cells. The G1 and G2/M populations correspond to cells with 2N and 4N DNA content respectively. c. Western blot analysis of transduced RPE-1 cells shown in (a). Samples labeled “NT” are the no treatment control. “PT” indicates the pre-treatment sample and “hpt” shows the time post-treatment in hours. The GFP blot shows the relative protein VII-GFP and GFP expression. H3S10ph indicates mitotic chromosomes. Total histone H3 and vinculin loading controls are also shown. d. HEK 293T proliferation with and without protein VII-HA expression. The protein VII-HA + expression and GFP cells were induced to express the target proteins under a doxycycline inducible promoter for 96 hours. The protein VII-HA no expression cells were not treated with doxycycline. At 96 hours post-treatment, there were significantly fewer cells expressing protein VII-HA compared to the GFP control and the untreated protein VII-HA cells (one-way ANOVA with multiple comparisons; p =0.0016 and p =0.0009 respectively). There was no significant difference in cell number between the GFP control and untreated protein VII-HA cells ( p =0.7841). Error bars represent SD. N = 3. e. Flow cytometry cell cycle analysis of HEK 293T cells shown in (d) using DNA content to assess cell cycle progression as described (b). Since more than 90% of the doxycycline treated GFP cells were positive for GFP expression, the cells were not gated on positive expression. DNA content is shown on the x-axis and G1 and G2/M peaks are indicated although the ploidy of HEK 293T cells varies within the population of cells. The y-axis histogram is normalized to fit the entire G1 peak for each sample. f. Western blot analysis of HEK 293T cells shown in (d). HA- and GFP-probed blots indicate expression of target proteins in doxycycline-treated samples. E1A indicates the presence of adenovirus early expressed E1A protein which contributes to the transformation of HEK 293T cells. Multiple E1A isoforms are resolved as multiple bands. H3S10ph indicates mitotic chromatin. Total histone H3 and vinculin are loading controls. g. Model for protein VII on host chromatin during infection. An uninfected cell has normal cell cycle regulation. Upon infection, the early-expressed adenovirus genes E1A and E1B bypass cell cycle checkpoints and push the cell into an S phase-like gene expression program. E1A acts primarily through redistributing acetylation of histone H3K18 globally. Late during infection, abundant protein VII recruits HMGB1 and SET to displace the linker histone from host chromatin. Protein VII chromatin disruption impedes mitosis and therefore cytokinesis, culminating in cell lysis and maximizing the release of viral progeny.

Article Snippet: 10 uL of each sample was run on a 15% SDS PAGE gel using standard methods as described above with the following primary antibodies: α-vinculin (1:20,000 Sigma #V9131), α-GFP (1:2500, Abcam #290), α-H3 (1:20,000, Abcam #1791, α-H3S10ph (1:1000, EMD Millipore #06-570), α-HA (1:500, BioLegend/Covance #MMS-101R) or α-E1A (1:250, BD Biosciences #554155); and secondary antibodies: α-mouse (Jackson ImmunoResearch #115-035-003) or α-rabbit (Jackson ImmunoResearch #111-035-045).

Techniques: Transduction, Recombinant, Inhibition, Expressing, Standard Deviation, Cell Cycle Assay, Flow Cytometry, Staining, Western Blot, Labeling, Transformation Assay, Infection, Lysis

Journal: Cancer Science

Article Title: Angiotensin II subtype 1a receptor signaling in resident hepatic macrophages induces liver metastasis formation

doi: 10.1111/cas.13306

Figure Lengend Snippet: Angiotensin II (Ang II )/ AT 1a axis enhances transforming growth factor‐β1 ( TGF ‐β1) expression in Kupffer cells. (a) Accumulation of F4/80 + cells, TGF ‐β1 + cells, and F4/80 + / TGF ‐β1 + cells in metastatic areas from WT and AT 1a KO mice on day 14. Double‐staining of liver sections with antibodies against F4/80 (red) and TGF ‐β1 (green) in WT and AT 1a KO mice. Expression of TGF ‐β1 was colocalized with F4/80 + cells. Metastatic area is delineated with the white dashed line. T, tumor. Scale bar = 100 μm. (b–d) Numbers of F4/80 + cells (b), TGF ‐β1 + cells (c), and F4/80 + TGF ‐β1 + cells (d) in metastatic areas from WT and AT 1a KO mice. Data are expressed as the means ± SD of six mice per group. * P < 0.05. (e) Expression of AT 1a and AT 1b in KUP 5 Kupffer cells. Data are expressed as the means ± SD of six mice per group. (f) Expression of TGF ‐β1 on KUP 5 cells under stimulation with Ang II . Expression of TGF ‐β1 was enhanced 6 h after stimulation with Ang II compared with control. There was no significant difference at 12 h. Data are expressed as the means ± SD of six mice per group. * P < 0.05 versus control.

Article Snippet: For the latter, sections were activated using Histo VT One (Nacalai Tesque, Yokohama, Japan) and then incubated overnight at 4°C with one of the following primary antibodies: (a) anti‐mouse F4/80 antibody (1:200, rat monoclonal, sc52664; Santa Cruz Biotechnology, Dallas, TX, USA); (b) anti‐mouse TGF‐β1 antibody (1:200, rabbit polyclonal, ab92486; Abcam, Cambridge, UK); (c) anti‐mouse GFP antibody (1:200, rabbit polyclonal, ab290; Abcam); (d) anti‐mouse Ang II type 1A receptor antibody (1:100, rabbit polyclonal, bs‐2132R; Bioss, Boston, MA, USA); (e) anti‐mouse desmin antibody (1:100, goat polyclonal, ab80503; Abcam); (f) anti‐mouse type I collagen antibody (1:100, rabbit polyclonal, ab21286; Abcam); or (g) anti‐mouse CD31 antibody (1:200, rabbit polyclonal, ab28364; Abcam).

Techniques: Expressing, Double Staining