Journal: Cancer Immunology, Immunotherapy : CII

Article Title: miR-375 inhibits Helicobacter pylori -induced gastric carcinogenesis by blocking JAK2–STAT3 signaling

doi: 10.1007/s00262-014-1550-y

Figure Lengend Snippet: The regulation of JAK2–STAT3 signaling pathway is involved in H. pylori-induced cell proliferation and migration by activating the downstream target genes BCL-2 and TWIST1. a, b qRT-PCR results of BCL-2 and TWIST1 mRNA in BGC-823 (a) and GES-1 (b) cells after JAK2 silencing and H. pylori infection for 12 h; GAPDH was used as an internal control. The results were normalized to the mRNA level transfected with negative control of siRNAs. c, d Western blotting and quantitative analysis of BCL-2 and TWIST1 expression in BGC-823 (c) and GES-1 (d) cells after JAK2 silencing and H. pylori infection for 24 h; β-actin expression was used as an internal control. e, f CCK-8 results of BGC-823 (e) and GES-1 (f) cells after BCL-2 silencing and H. pylori infection for the indicated times. g, h The migratory ability of BGC-823 (g) and GES-1 (h) cells was evaluated by transwell assays after TWIST1 silencing and H. pylori infection for 12 h. Representative image fields of the migratory cells on the membrane stained with crystal violet (×200), and OD570 of crystal violet stain was determined to quantify the transwell assays. The data are represented as the mean ± SD of triplicate samples, *P < 0.05, **P < 0.01 versus the control group

Article Snippet: The membranes were blocked overnight with 5 % nonfat milk at 4 °C and incubated with anti-JAK2 RabMab (1:1,000, Cell Signaling Technology, CST), anti-phospho-JAK2 (Tyr1007/1008) RabMab (1:1,000, CST), anti-STAT3 RabMab (1:1,000, CST), anti-phospho-STAT3 (Tyr705) RabMab (1:2,000, CST), anti-BCL-2 rabbit polyconal Ab (1: 750, Signalway Antibody, SAB), or anti-TWIST1 rabbit polyconal Ab (1:750, SAB) as indicated for 12–16 h at 4 °C or with anti-β-actin monoclonal antibody (1:500, ZSGB-Bio) as a control.

Techniques: Migration, Quantitative RT-PCR, Infection, Transfection, Negative Control, Western Blot, Expressing, CCK-8 Assay, Membrane, Staining

Journal: Blood Advances

Article Title: ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells

doi: 10.1182/bloodadvances.2020002395

Figure Lengend Snippet: ICOS expression by malignant T cells in CTCL skin biopsy specimens at different disease stages. (A) Percentage of patients with ICOS+ tumoral cells, determined by immunohistochemical analysis of ICOS expression in skin samples of 23 patients with early-stage MF, 12 with transformed MF, 17 with SS, 12 with B-cell lymphoma, 14 with CD30+ LPD (cutaneous anaplastic large cell lymphoma and lymphomatoid papulosis), 12 with PCSMTLPD, and 13 with AITL. Cutaneous B-cell lymphoma skin samples were used as negative controls, whereas PCSMTLPD and AITL were positive controls. (B) Immunohistochemical analysis of the expression of ICOS in epidermal or dermal CD3+CD4+ infiltrating lymphocytes. (C) Double-staining experiments using immunofluorescence with anti-mouse Texas red anti-rabbit Alexa Fluor 488 as secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear staining in skin (line 1) and lymph node samples (lines 2 and 3) from 3 different patients with SS. Arrowheads identify cells with a single-marker expression; green arrowheads show ICOS+ cells, and red arrowheads show CD8+ cells (line 2) and CD4+ cells (line 3). (D) Double-staining immunohistochemical analysis of skin and node samples of 5 patients with SS. The percentage of patients with ICOS+/CD4+, ICOS+/CD8+, ICOS+/FoxP3+, and ICOS+/PD-1+ cells in the skin and node tumoral infiltrate is represented. cALCL, cutaneous anaplastic large cell lymphoma.

Article Snippet: 28 Flow cytometry and immunochemistry We used rabbit anti-ICOS antibodies (rabbit polyconal antibody from Spring Biosciences [Abcam, Cambridge, United Kingdom] for immunohistochemistry and SP98 rabbit mAb from Spring Biosciences, with anti-rabbit Alexa488 secondary antibodies), as well as mouse antibodies to PD-1 (NAT105, Abcam), CD4 (4B12, Novocastra) (Leica Biosystems, Wetzlar, Germany), CD8 (C8/144B, Dako) (Agilent Technologies, Santa Clara, CA), and FoxP3 (236A/E7, Abcam) for fluorescent multiplex staining using anti-mouse Texas red secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear staining.

Techniques: Expressing, Immunohistochemical staining, Transformation Assay, Double Staining, Immunofluorescence, Staining, Marker

Journal: Blood Advances

Article Title: ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells

doi: 10.1182/bloodadvances.2020002395

Figure Lengend Snippet: ICOS expression of malignant cells in the blood of patients with SS. (A) Percentages of lymphoid cell populations within peripheral blood of 13 patients with SS and 12 healthy donors using flow cytometry. NK, natural killer. (B) Gating strategy for Sézary cells (CD4+CD158k+CD7−) by flow cytometry for a representative patient. (C) Flow cytometric evaluation of ICOS expression in patients with SS and healthy donors. Percentage of ICOS+ cells among Sézary CD4+ T cells and nontumoral CD4+ T cells. Sézary cells were defined as CD4+, KIR3DL2+, CD7−, or CD4+, KIR3DL2+, CD26−. Nontumoral CD4+ T cells are KIR3DL2−. Approximately 70% of Sézary cells expressed ICOS compared with <40% of nontumoral CD4+ cells in patients and CD4+ cells in healthy donors. (D) Gating strategy for Treg cells (CD4+FoxP3+CD25+) by flow cytometry for a representative healthy donor and patient. (E) Percentage of ICOS+ Treg cells in patients with SS and healthy donors. ***P < .001; **P =.001-.01; *P = .01-.05. IC, isotypic control.

Article Snippet: 28 Flow cytometry and immunochemistry We used rabbit anti-ICOS antibodies (rabbit polyconal antibody from Spring Biosciences [Abcam, Cambridge, United Kingdom] for immunohistochemistry and SP98 rabbit mAb from Spring Biosciences, with anti-rabbit Alexa488 secondary antibodies), as well as mouse antibodies to PD-1 (NAT105, Abcam), CD4 (4B12, Novocastra) (Leica Biosystems, Wetzlar, Germany), CD8 (C8/144B, Dako) (Agilent Technologies, Santa Clara, CA), and FoxP3 (236A/E7, Abcam) for fluorescent multiplex staining using anti-mouse Texas red secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear staining.

Techniques: Expressing, Flow Cytometry

Journal: Blood Advances

Article Title: ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells

doi: 10.1182/bloodadvances.2020002395

Figure Lengend Snippet: Anti-ICOS ADCs have a specific in vitro efficacy in ICOS-expressing cell lines. (A) Immunophenotyping of CTCL cell lines using flow cytometry. Note that ICOS expression was higher in MyLa and MJ cells than in HUT78 cells. (B-F) Percentage of cell viability in increasing ADC concentrations, assessed with alamarBlue (mean of 16 replicates), on MyLa cells (B), MJ cells (C), HUT78 cells (D), Jurkat cells (E), and Jurkat-ICOS cells (F). Anti-HER2 ADCs were used as a negative control, whereas anti-CD30 ADCs (BV) were a positive control. (G) Summary table of IC50 values expressed in ng/mL of all ADCs. ***P < .001. ns, not significant. PBD, pyrrolobenzodiazepine.

Article Snippet: 28 Flow cytometry and immunochemistry We used rabbit anti-ICOS antibodies (rabbit polyconal antibody from Spring Biosciences [Abcam, Cambridge, United Kingdom] for immunohistochemistry and SP98 rabbit mAb from Spring Biosciences, with anti-rabbit Alexa488 secondary antibodies), as well as mouse antibodies to PD-1 (NAT105, Abcam), CD4 (4B12, Novocastra) (Leica Biosystems, Wetzlar, Germany), CD8 (C8/144B, Dako) (Agilent Technologies, Santa Clara, CA), and FoxP3 (236A/E7, Abcam) for fluorescent multiplex staining using anti-mouse Texas red secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear staining.

Techniques: In Vitro, Expressing, Flow Cytometry, Negative Control, Positive Control

Journal: Blood Advances

Article Title: ICOS is widely expressed in cutaneous T-cell lymphoma, and its targeting promotes potent killing of malignant cells

doi: 10.1182/bloodadvances.2020002395

Figure Lengend Snippet: In vivo efficacy of anti-ICOS-MMAE ADCs on ICOS+PDXs. (A-C) Fourteen mice were engrafted with 5.105 cells of PDXs from patients with SS and divided into 2 groups (anti-ICOS-MMAE ADC and anti-HER2 ADC control). Both treatments were injected IV at days 55, 58, 62, and 65 at a dose of 3 mg/kg. Mice were then euthanized at day 69, and organs were removed, dissociated, and analyzed by flow cytometry (in the blood [A], bone marrow [B], and spleen [C]). (D) Thirty mice were engrafted with 5.105 cells of PDXs from patients with AITL and divided into 3 groups of 10 mice. Treatment began on day 22, when the earliest blasts were detected in the blood (∼0.2 blasts/μL). Anti-ICOS ADC and saline serum (NaCl 0.9%) were injected IV at days 22, 25, 38, and 43 at a dose of 3 mg/kg. Vincristine was administered intraperitoneally on days 22, 29, and 38 at 0.25 mg/kg. *P = .01-.05; ***P < .001.

Article Snippet: 28 Flow cytometry and immunochemistry We used rabbit anti-ICOS antibodies (rabbit polyconal antibody from Spring Biosciences [Abcam, Cambridge, United Kingdom] for immunohistochemistry and SP98 rabbit mAb from Spring Biosciences, with anti-rabbit Alexa488 secondary antibodies), as well as mouse antibodies to PD-1 (NAT105, Abcam), CD4 (4B12, Novocastra) (Leica Biosystems, Wetzlar, Germany), CD8 (C8/144B, Dako) (Agilent Technologies, Santa Clara, CA), and FoxP3 (236A/E7, Abcam) for fluorescent multiplex staining using anti-mouse Texas red secondary antibodies and 4′,6-diamidino-2-phenylindole for nuclear staining.

Techniques: In Vivo, Injection, Flow Cytometry

Journal: Cell metabolism

Article Title: Abrogating mitochondrial dynamics in mouse hearts accelerates mitochondrial senescence

doi: 10.1016/j.cmet.2017.09.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyconal anti-LC3 A/B , Cell Signaling Technology , Cat# 4108.

Techniques: Virus, Plasmid Preparation, Recombinant, TUNEL Assay, Software