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

Journal:

Article Title: Human Herpesvirus 6 Activates Lytic Cycle Replication of Kaposi's Sarcoma-Associated Herpesvirus

doi:

Figure Lengend Snippet: Northern blot analysis for ORF26 mRNA in BCBL-1 cells. A: ORF26 mRNA expressed in BCBL-1 cells after co-culture with HHV-6-infected JJhan cells by direct cell-cell contact. Total RNA isolated from BCBL-1 cells treated with TPA for 48 hours (positive control, BCBL-1 + TPA 48 hours), BCBL-1 cells co-cultured with HHV-6-infected JJhan cells (JJhan + HHV-6), 8, 24, 72, and 96 hours, respectively (+JJhan + HHV-6 8 hours, 24 hours, 72 hours, 96 hours), BCBL-1 cells co-cultured with uninfected JJhan cells, 8, 24, 72, and 96 hours, respectively (+JJhan 8 hours, 24 hours, 72 hours, 96 hours), BCBL-1 cells alone (negative control, BCBL-1 alone) were transferred to nylon membrane and ORF26 mRNA expression was detected by Northern blot. The same membrane was stripped and reprobed for GAPDH to demonstrate equal loading of the RNA. B: ORF26 mRNA expressed in BCBL-1 cells cultured for 24 to 96 hours in 50% conditioned medium. ORF26 mRNA expression in BCBL-1 cells cultured in 50% conditioned medium from HHV-6-infected JJhan cells for 24, 48, and 96 hours (+JJhan + HHV-6 24 hours, 48 hours, 96 hours) and BCBL-1 cells cultured in 50% conditioned medium from uninfected JJhan cells for 24, 48, and 96 hours (+JJhan 24 hours, 48 hours, 96 hours) was detected by Northern blot. C: ORF26 mRNA expressed in BCBL-1 cells cultured for 24 to 96 hours in KSHV-depleted conditioned medium. ORF26 mRNA expression in BCBL-1 cells cultured in KSHV-depleted conditioned medium for 24, 48, 72, and 96 hours (24 hours, 48 hours, 72 hours, 96 hours) and normal BCBL-1 cells (control) was detected by Northern blot. All results shown are a representative experiment of at least three independent experiments with similar results.

Article Snippet: The panel of mAbs used included KSHV ORF59 (clone 11D1, mouse IgG 2b ; Advanced Biotechnologies Inc.) and KSHV ORF K8.1 A/B (clone 4A4, mouse IgG 1 ; Advanced Biotechnologies Inc.).

Techniques: Northern Blot, Co-Culture Assay, Infection, Isolation, Positive Control, Cell Culture, Negative Control, Membrane, Expressing, Control

Journal:

Article Title: Human Herpesvirus 6 Activates Lytic Cycle Replication of Kaposi's Sarcoma-Associated Herpesvirus

doi:

Figure Lengend Snippet: Infection of BCBL-1 cells with HHV-6 induces lytic cycle replication of KSHV. A: ORF26 mRNA expressed in BCBL-1 cells infected with pelleted HHV-6. ORF26 mRNA expression in BCBL-1 cells infected with pelleted HHV-6 for 16, 24, 48, 72, and 96 hours (16 hours, 24 hours, 48 hours, 72 hours, 96 hours), and uninfected BCBL-1 (control) cells was detected by Northern blot. A representative experiment is shown; at least three independent experiments were performed with similar results. B: Real-time quantitative PCR for ORF26 mRNA in BCBL-1 and BC-3 cells after infection with pelleted HHV-6. ORF26 mRNA expression in BCBL-1 (white bar) and BC-3 (black bar) cells infected with pelleted HHV-6 for 16, 24, 48, 72, 96, and 120 hours was quantitated by real-time quantitative PCR. Results shown were the statistic of three independent experiments performed in triplicate. C: Infection of HHV-6 promotes induction of KSHV ORF50 promoter activity. BCBL-1 (white bar) and BC-3 (black bar) cells were transfected with pGL-3 basic control or p50 plasmids. p50-transfected cells were either untreated (p50), treated with TPA (p50 + TPA), or infected with HHV-6 (p50 + HHV-6). Luciferase activities were measured as induction (n-fold). Results from a representative experiment performed in triplicate are shown; five independent experiments were performed with similar results.

Article Snippet: The panel of mAbs used included KSHV ORF59 (clone 11D1, mouse IgG 2b ; Advanced Biotechnologies Inc.) and KSHV ORF K8.1 A/B (clone 4A4, mouse IgG 1 ; Advanced Biotechnologies Inc.).

Techniques: Infection, Expressing, Control, Northern Blot, Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Luciferase

Journal:

Article Title: Human Herpesvirus 6 Activates Lytic Cycle Replication of Kaposi's Sarcoma-Associated Herpesvirus

doi:

Figure Lengend Snippet: Immunohistochemisty staining of BCBL-1 cells co-cultured with HHV-6-infected JJhan cells and infected with pelleted HHV-6. KSHV lytic proteins ORF K8.1 (top) and ORF59 (bottom) expression in JJhan cells, BCBL-1 cells, TPA-treated BCBL-1 cells (BCBL-1 + TPA), PBS-treated BCBL-1 cells (BCBL-1 + PBS), BCBL-1 cell co-cultured with JJhan cells (BCBL-1/JJhan), BCBL-1 cell co-cultured with HHV-6-infected JJhan cells (BCBL-1/JJhan + HHV-6), or HHV-6 infected BCBL-1 cells (BCBL-1 + HHV-6) were detected by immunohistochemistry with ORF K8.1 and ORF59 monoclonal antibodies. * and **, Statistically significant increases in ORF K8.1 and ORF59 expression compared to that of BCBL-1 cells co-cultured with normal JJhan cells for 3 days and cultured in PBS buffer for 6 days, respectively. Original magnifications, ×60.

Article Snippet: The panel of mAbs used included KSHV ORF59 (clone 11D1, mouse IgG 2b ; Advanced Biotechnologies Inc.) and KSHV ORF K8.1 A/B (clone 4A4, mouse IgG 1 ; Advanced Biotechnologies Inc.).

Techniques: Staining, Cell Culture, Infection, Expressing, Immunohistochemistry, Bioprocessing

Journal:

Article Title: Human Herpesvirus 6 Activates Lytic Cycle Replication of Kaposi's Sarcoma-Associated Herpesvirus

doi:

Figure Lengend Snippet: IFN-γ partially contributes to induction of KSHV replication. A: Expression of IFN-γ in BCBL-1 cells co-cultured for 8 to 120 hours with HHV-6-infected JJhan cells and cultured in 50% conditioned medium. Supernatants from BCBL-1 cells co-cultured with JJhan (JJhan + BCBL-1), HHV-6-infected JJhan [JJhan(HHV-6) + BCBL-1] or BCBL-1 cells cultured in conditioned medium from JJhan cell culture (SuJJhan + BCBL-1) and HHV-6-infected JJhan cell culture [SuJJhan(HHV-6) + BCBL-1] for various times indicated were collected for detection of IFN-γ by ELISA. Results were the statistic of three independent experiments with duplicate. B: Real-time quantitative PCR analysis for ORF26 mRNA expression in blocking assay by mAb against IFN-γ. Real-time quantitative PCR was used to detected relative quantities of ORF26 mRNA in normal BCBL-1 cells (BCBL-1 alone), IFN-γ-treated BCBL-1 cells (IFN-γ + BCBL-1), BCBL-1 cells co-cultured with JJhan cell (JJhan + BCBL-1), HHV-6-infected JJhan cells [JJhan(HHV-6) + BCBL-1], or HHV-6-infected JJhan cells plus mAb treatment [JJhan(HHV-6) + BCBL-1 + mAb] for various times indicated. The ORF26 mRNA expression in BCBL-1 cells cultured in conditioned medium from JJhan cell (SuJJhan + BCBL-1), HHV-6-infected JJhan cells [SuJJhan(HHV-6) + BCBL-1], or HHV-6-infected JJhan cells plus mAb treatment [SuJJhan(HHV-6) + BCBL-1 + mAb] for various times indicated was also detected by real-time quantitative PCR. The statistic of three independent experiments performed in triplicate was shown. C: Northern blot analysis for ORF26 mRNA expression in blocking assay by mAb against IFN-γ. Northern blot analysis was used to detect ORF26 mRNA in BCBL-1 cells co-cultured with HHV-6-infected JJhan cells at 24 and 48 hours or cultured in conditioned medium at 48 and 72 hours before or after treatment with mAb against IFN-γ, respectively. Lane 1 (from left), BCBL-1 co-cultured with HHV-6-infected JJhan cells (BCBL-1 + JJhan + HHV-6), 24 hours; lane 2, BCBL-1 co-cultured with HHV-6-infected JJhan cells with addition of mAb against IFN-γ (BCBL-1 + JJhan + HHV-6 + mAb), 24 hours; lane 3, BCBL-1 + JJhan + HHV-6, 48 hours; lane 4, BCBL-1 + JJhan + HHV-6 + mAb, 48 hours; lane 5, BCBL-1 cells cultured in conditioned medium from HHV-6-infected JJhan cells with addition of mAb against IFN-γ (BCBL-1 + C.M. + mAb), 72 hours; lane 6, BCBL-1 + C.M., 72 hours; lane 7, BCBL-1 + C.M. + mAb, 48 hours; lane 8, BCBL-1 + C.M., 48 hours. A representative experiment is shown; three independent experiments were run and gave similar results.

Article Snippet: The panel of mAbs used included KSHV ORF59 (clone 11D1, mouse IgG 2b ; Advanced Biotechnologies Inc.) and KSHV ORF K8.1 A/B (clone 4A4, mouse IgG 1 ; Advanced Biotechnologies Inc.).

Techniques: Expressing, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Blocking Assay, Northern Blot

Journal: Cancer Medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma

doi: 10.1002/cam4.70190

Figure Lengend Snippet: The effect of SUZ12 on metastasis related genes expression was tested by qRT‐PCR and western blotting. sh‐SUZ12 increased MMP1/2/3/9 mRNA expression (A) and TIMP1/2 protein expression (B), while decreased MMP14 mRNA expression (A), MMP1/9/14 (B), TIMP3 (B), and ITGB1/5(F) protein expression, without significantly effected ITGBs mRNA expression (E). oe‐SUZ12 increased MMP14 mRNA (C) and protein (D) expression, while decreased TIMP2 mRNA expression (C) and TIMP1/2 (D) protein expression.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122‐1‐AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103‐1‐AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052‐1‐AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939‐1‐AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554‐1‐AP), p18 (1:1000, BOSTER China, cat. no. M03299‐1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283‐2‐Ig), p‐p53 (1:2000; Proteintech, USA, cat. no. 28961‐1‐AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl‐2 (1:1000; Proteintech, USA, cat. no. 26593‐1‐AP), Bax (1:2000; Proteintech, USA, cat. no. 50599‐2‐lg), E‐cadherin (1:5000; Proteintech, USA, cat. no. 20874‐1‐AP), N‐cadherin (1:3000; Proteintech, USA, cat. no. 22018‐1‐AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366‐1‐AP), MMP1 (1:1000, BOSTER China, cat. no. A00733‐1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs‐0415R), TIMP2 (1:1000, Bioss China, cat. no. bs‐10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858‐1‐AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201‐1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD‐L1 (1:3000; Proteintech, USA, cat. no. 66248‐1‐Ig), and β‐actin (1:5000; Proteintech, USA, cat. no. 66009‐1‐Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot