Journal: Oncology Letters

Article Title: Simian virus 40 may be associated with developing malignant pleural mesothelioma

doi: 10.3892/ol.2016.4174

Figure Lengend Snippet: Expression of simian virus 40 large T antigen was assessed by immunohistochemical staining using the pAb101 antibody. The nuclei of the tumor cells are strongly immunoreactive (brown color; original magnification, ×100). The specimen in the present figure was provided an Allred score of 3 + .

Article Snippet: The Lab Vision mouse monoclonal antibody pAb101 (dilution, 1:100; catalog no., MS-1832-P; Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to detect SV40 Tag expression.

Techniques: Expressing, Virus, Immunohistochemical staining, Staining

Journal: mBio

Article Title: Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

doi: 10.1128/mBio.00620-12

Figure Lengend Snippet: Inflammasome components equip macrophages to disrupt L. pneumophila replication vacuoles, which correlates with autophagy. (A) After infection of C57BL/6J macrophages with WT or flaA mutant L. pneumophila at an MOI of <1 for 18 or 40 h, bacteria (red) were vacuolar ( flaA- 18h), dispersed rods (WT-18h) or degraded (WT-40 h). (B) The mean (± SE) percentage of macrophages containing dispersed WT or flaA mutant bacteria 18 h after infection was calculated for >100 infected macrophages of the genotypes indicated in three (WT) or two ( flaA ) experiments. *, P < 0.05 compared to C57BL/6J cells infected with WT bacteria. (C) After infection of macrophages from transgenic LC3-GFP C57BL/6J mice for 13 h with WT or flaA mutant Legionella (red) at an MOI of <1, the mean % (± SE) of macrophages of each of four morphological classes (blue nuclei) that contained at least 3 LC3-GFP+ puncta (green) was calculated by scoring >200 macrophages in each of four experiments. **, P < 0.01 and *, P < 0.05, compared to macrophages with normal nuclei and vacuolar bacteria. Arrows indicate LC3 colocalization with bacteria. (D) After infecting C57BL/6J macrophages for 13 h with WT or flaA mutant L. pneumophila at an MOI of <1, the mean (± SE) % uninfected (RPMI) or infected macrophages (red) containing two or more p62 puncta (green) was calculated by scoring at least 100 macrophages in three (RPMI) or four (WT, flaA ) independent experiments. **, P < 0.01 and *, P < 0.05 compared to uninfected macrophages.

Article Snippet: Next cells were infected with WT, dotA , or flaA mutant L. pneumophila at an MOI of <3 in Hanks buffer for 15 min. Macrophages were then fixed with PLP-sucrose and permeabilized with methanol, and endogenous LC3 was stained using mouse anti-LC3 antibody (1:100; MBL) and Oregon green-conjugated anti-mouse IgG antibodies (1:150, Invitrogen) as described previously ( ).

Techniques: Infection, Mutagenesis, Transgenic Assay

Journal: mBio

Article Title: Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

doi: 10.1128/mBio.00620-12

Figure Lengend Snippet: Inflammasome components promote rapid autophagosome flux in response to flagellate, type IV secretion-competent L. pneumophila . (A) Transgenic GFP-LC3 C57BL/6J macrophages were maintained in RPMI or treated for 20 min with Hanks buffer before identifying autophagosomes using GFP-specific antibody (LC3). Alternatively, after a population of autophagosomes had been generated, macrophages were infected for 10 min with WT, flaA , or dotA mutant L. pneumophila at an MOI of 3 (WT, dotA ) or <10 ( flaA ) before immunolabeling of bacteria (Lp) and autophagosomes (LC3). Arrowheads indicate infected macrophages. (B) Macrophages of the indicated genotypes were treated as described above to calculate the mean (± SE) percentage of 100 macrophages containing >3 autophagosomes labeled with LC3-specific antibody in each of three experiments. **, P < 0.01; *, P = 0.05, compared to Hanks buffer-treated cells; #, P = 0.04 compared to C57BL/6J macrophages treated with Hanks buffer. (C) C57BL/6 transgenic GFP-LC3 macrophages were incubated for 30 min with or without 25 nM bafilomycin A, an inhibitor of autophagosome-lysosome fusion, treated for 20 min with Hanks buffer with or without bafilomycin A, and then incubated for an additional 15 min without or with L. pneumophila at an MOI of ~1. More than 100 macrophages were scored for the presence of >3 LC3 vacuoles localized using GFP-specific antibody. Means (± SE) were calculated from four experiments. **, P < 0.05 compared with corresponding Hanks buffer-treated macrophages; #, P < 0.01 compared with macrophages treated with Hanks buffer but not bafilomycin A. (D) Macrophages of the genotypes shown were treated with Hanks buffer for 30 min, and then autophagosomes were visualized using LC3-specific antibody.

Article Snippet: Next cells were infected with WT, dotA , or flaA mutant L. pneumophila at an MOI of <3 in Hanks buffer for 15 min. Macrophages were then fixed with PLP-sucrose and permeabilized with methanol, and endogenous LC3 was stained using mouse anti-LC3 antibody (1:100; MBL) and Oregon green-conjugated anti-mouse IgG antibodies (1:150, Invitrogen) as described previously ( ).

Techniques: Transgenic Assay, Generated, Infection, Mutagenesis, Immunolabeling, Labeling, Incubation

Journal: mBio

Article Title: Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

doi: 10.1128/mBio.00620-12

Figure Lengend Snippet: Caspase-1 promotes autophagy in response to K + efflux. (A) Caspase-1 cleavage by macrophages treated for 1 h with valinomycin or 2 h with 20 µM nigericin. Results are representative of two experiments. (B) GFP-LC3 + autophagosomes of transgenic C57BL/6J macrophages maintained in RPMI or exposed 1 h to valinomycin with or without 150 mM KCl. (C) Mean (± SE) percentage of macrophages containing >3 GFP-LC3 + vacuoles after 1 h with 50 µM valinomycin with or without 150 mM KCl and with or without 1 h pretreatment with 100 µM YVAD, a caspase-1 inhibitor, was calculated for >100 cells in each of >3 experiments. **, P < 0.01 compared to valinomycin-treated macrophages. (D) Lipidated GFP-LC3II in transgenic macrophages in RPMI or treated for 0.5 to 2 h with 100 µM valinomycin with or without KCl and with or without 1 h YVAD pretreatment. Similar results were obtained in three other experiments. (E) Pyroptosis by macrophages cultured for 1 h with or without 100 µM YVAD and incubated for 1 h without (RPMI) or with WT L. pneumophila at an MOI ~50 was measured as the mean percentage (± SD) of >100 macrophages whose nucleus was round and phase dense or as the percentage of LDH released from macrophages with or without YVAD pretreatment and 1 h incubation with WT or flaA mutant L. pneumophila at an MOI of ~25. The mean (± SD) percenage of LDH released in the absence of flagellin or presence of YVAD was calculated relative to LDH released after infection with WT bacteria calculated in two experiments. **, P < 0.01 compared infection with WT. (F) Lipidated LC3II and actin in macrophages of the genotypes shown treated for 0 to 60 min with 50 µM valinomycin. Similar results were obtained in three experiments.

Article Snippet: Next cells were infected with WT, dotA , or flaA mutant L. pneumophila at an MOI of <3 in Hanks buffer for 15 min. Macrophages were then fixed with PLP-sucrose and permeabilized with methanol, and endogenous LC3 was stained using mouse anti-LC3 antibody (1:100; MBL) and Oregon green-conjugated anti-mouse IgG antibodies (1:150, Invitrogen) as described previously ( ).

Techniques: Transgenic Assay, Cell Culture, Incubation, Mutagenesis, Infection

Journal: mBio

Article Title: Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

doi: 10.1128/mBio.00620-12

Figure Lengend Snippet: Autophagy protects macrophages from pyroptosis induced by L. pneumophila . (A) C57BL/6J macrophages cultured 1 h with or without 50 µM Atg4 inhibitor or 10 mM 3-MA were then incubated 35 min in rich medium (RPMI) or amino acid-free Hanks buffer with or without inhibitors before immunolocalization of autophagosomes. The mean fraction (± SE) of macrophages that contained >3 LC3 puncta was calculated by scoring >100 macrophages in each of two or three samples in two independent experiments. Similar results were obtained when the Atg4 inhibitor was analyzed in two other independent experiments that analyzed either endogenous LC3 or GFP-LC3. **, P < 0.01 compared to macrophages exposed only to Hanks buffer. (B) C57BL/6J macrophages cultured for 1 h with or without 50 µM Atg4 inhibitor or 10 mM 3-MA were then incubated 30 min in RPMI or Hanks buffer with or without inhibitors before analysis of endogenous LC3 protein. Similar patterns were observed in three other independent experiments. (C) After infecting macrophages from transgenic LC3-GFP C57BL/6J mice for 2 h with WT Legionella , cells were cultured for an additional 11 h with or without 50 µM Atg4 inhibitor before the mean percentage (± SE) of macrophages that contained at least 3 LC3-GFP+ puncta was calculated. In each of two experiments (Exp 1 and Exp 2), >100 total macrophages on 2 to 4 coverslips were scored. #, P = 0.08; **, P < 0.01, compared to infected but untreated cultures. (D) C57BL/6J macrophages cultured 1 h with or without inhibitors of Atg4 (50 µM) or caspase-1 (100 µM YVAD) were then infected with L. pneumophila with or without inhibitors for 2 h before cells were fixed and analyzed. The mean fraction (± SE) of macrophages containing the indicated number of bacteria whose nucleus was condensed was calculated by scoring >50 infected macrophages in each of 5 to 6 samples in one experiment. Similar results were obtained in three other independent experiments. **, P < 0.01 compared to macrophages that were untreated (RPMI) or treated with each or both inhibitors.

Article Snippet: Next cells were infected with WT, dotA , or flaA mutant L. pneumophila at an MOI of <3 in Hanks buffer for 15 min. Macrophages were then fixed with PLP-sucrose and permeabilized with methanol, and endogenous LC3 was stained using mouse anti-LC3 antibody (1:100; MBL) and Oregon green-conjugated anti-mouse IgG antibodies (1:150, Invitrogen) as described previously ( ).

Techniques: Cell Culture, Incubation, Transgenic Assay, Infection

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