Journal: Scientific Reports

Article Title: Phytocyanin-encoding genes confer enhanced ozone tolerance in Arabidopsis thaliana

doi: 10.1038/s41598-022-25706-0

Figure Lengend Snippet: Cellular localisation and the ability of AtUC5-GFP fusion proteins to confer O 3 tolerance. ( a ) Diagrams depicting the chimeric genes to produce fusion proteins in transgenic plants. ( b ) Results of western blotting to detect fusion protein expression in extracts of transgenic plants. Equal amounts (3 µg) of protein extracted from plants of various lines were subjected to electrophoresis and probed with anti-GFP antibody using Simple Western™ (Bio-Techne, Minneapolis, USA). GFP was added to the protein extract from the wild-type plant just before the electrophoresis for the lane “wt + GFP.” Results were obtained as digital data for each sample and exhibited as band images as their full-length blots. ( c ) Microscopic observation of GFP fluorescence in epidermal tissues of transgenic plants. α fluorescence images of epidermal tissue of a leaf transgenic for 35S:GFP-AtUC5 . β fluorescence image of epidermal tissue of a leaf transgenic for 35S:AtUC5-GFP . γ fluorescence image of epidermal tissue of a leaf transgenic for 35S:GFP-AtUC5 after treatment with hyperosmotic solution. δ fluorescence image of the epidermal tissue of a leaf transgenic for 35S:AtUC5-GFP after treatment with hyperosmotic solution. Arrowheads and arrows denote stomata and apoplastic matrices, respectively. ( d ) Diagrams depicting the presumed structures and localizations of fusion proteins in transgenic plants. In 35S:GFP-AtUC5 plants, the fusion protein is released from the plasma membrane after digestion at the GPI moiety by phospholipases. ( e ) Images of various lines 1 day after 2 h exposure to fresh air or 0.3 µL L −1 O 3 under 420 µmol photons m −2 s −1 illumination. Wt wild-type plants, GFP green fluorescent protein, 35S:GFP-AtUC5 35S:AtUC5-GFP , transgenic plants producing fusion proteins, 35S cauliflower mosaic virus 35S promoter, N a fragment encoding the N-terminal signal peptide of AtUC5, PLD plastocyanin-like domain, ALR arabinogalactan protein-like region, GPI glycosylphosphatidylinositol moiety, CGAS C-terminal glycosylphosphatidylinositol-anchored signal, FA fresh air .

Article Snippet: Equal amounts (3 µg) of protein extracted from plants of various lines were subjected to electrophoresis and probed with anti-GFP antibody using Simple Western™ (Bio-Techne, Minneapolis, USA).

Techniques: Transgenic Assay, Western Blot, Expressing, Electrophoresis, Fluorescence, Membrane, Virus

Journal: Cell reports

Article Title: The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases

doi: 10.1016/j.celrep.2023.113581

Figure Lengend Snippet: (A and B) HEK293T cells stably expressing ΔCARD Dmrb-CASP11 (A) or 2xFLAG-CASP11 (B) were transiently transfected with the indicated constructs. After 24 h, samples were treated with AP20187 (1 μM) (A) or LPS (25 μg/mL) (B) for 24 h and then analyzed for LDH release and immunoblotting. (C and E) Raw 264.7 macrophages were primed with LPS (5 μg/mL) for 4 h and then transfected with LPS (25 μg/mL) for 6 h before LDH release and immunoblot analysis (C) and IL-1 receptor activation by IL-1β (E). (D) Raw 264.7 macrophages were treated as in (C) and then analyzed for IL-1β release by ELISA. (F) HEK293T cell lysates expressing either human IL-18 or mouse IL-18 were incubated with one activity unit of the indicated recombinant caspases for 1 h or 24 h and then analyzed by immunoblotting. Data are means ± SEM of three biological replicates. ***p < 0.001, **p < 0.01, and *p < 0.05 by two-sided Student’s t test. Data are representative of three or more independent experiments. * = non-specific bands. The small m or h represents mouse or human proteins, respectively.

Article Snippet: Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), FLAG M2 monoclonal Ab (Sigma, F3165), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), CASP1 p20 Rabbit polyclonal Ab (Cell Signaling Tech, 2225s), CASP1 p12/10 Rabbit monoclonal Ab (Abcam, ab179515), CASP4 Rabbit polyclonal Ab (Cell Signaling Tech, 4450S), CASP5 Rabbit monoclonal Ab (Cell Signaling Tech, 46680S), Myc Mouse monoclonal Ab (Cell Signaling Tech, 2276S), V5 Rabbit monoclonal Ab (Cell Signaling Tech, 13202S), hIL-1β Goat Polyclonal Ab (R&D systems, AF-201-NA), hIL-18 Goat Ab (R&D systems, af2548), hIL-1α Recombinant Ab (PeproTech, 200-01A), PARP Rabbit polyclonal Ab (Cell Signaling Tech, 9542S), HA Rabbit monoclonal Ab (Cell Signaling Tech, 3724S), mIL-1β Goat polyclonal Ab (R&D systems, AF-401-NA), CASP11 Rat monoclonal Ab (Novus Biologicals, NB120-10454), FKBP12 Rabbit polyclonal Ab (Abcam, ab24373).

Techniques: Stable Transfection, Expressing, Transfection, Construct, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activity Assay, Recombinant

Journal: Cell reports

Article Title: The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases

doi: 10.1016/j.celrep.2023.113581

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), FLAG M2 monoclonal Ab (Sigma, F3165), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), CASP1 p20 Rabbit polyclonal Ab (Cell Signaling Tech, 2225s), CASP1 p12/10 Rabbit monoclonal Ab (Abcam, ab179515), CASP4 Rabbit polyclonal Ab (Cell Signaling Tech, 4450S), CASP5 Rabbit monoclonal Ab (Cell Signaling Tech, 46680S), Myc Mouse monoclonal Ab (Cell Signaling Tech, 2276S), V5 Rabbit monoclonal Ab (Cell Signaling Tech, 13202S), hIL-1β Goat Polyclonal Ab (R&D systems, AF-201-NA), hIL-18 Goat Ab (R&D systems, af2548), hIL-1α Recombinant Ab (PeproTech, 200-01A), PARP Rabbit polyclonal Ab (Cell Signaling Tech, 9542S), HA Rabbit monoclonal Ab (Cell Signaling Tech, 3724S), mIL-1β Goat polyclonal Ab (R&D systems, AF-401-NA), CASP11 Rat monoclonal Ab (Novus Biologicals, NB120-10454), FKBP12 Rabbit polyclonal Ab (Abcam, ab24373).

Techniques: Recombinant, Virus, CyQUANT Assay, LDH Cytotoxicity Assay, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

doi: 10.1136/jitc-2021-003237

Figure Lengend Snippet: Antibodies used for flow cytometry

Article Snippet: 1 , Anti-MUC1 (Novus Biologicals) , SM3 , AF700.

Techniques: Expressing, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

doi: 10.1136/jitc-2021-003237

Figure Lengend Snippet: The novel TR2.41BB costimulatory receptor induces 41BB signaling on TR2 engagement. (A) Mucin 1 (MUC1) expression on triple negative breast cancer (TNBC) cell lines BT-20, MDA-MB-231, and the MUC1- cell line 293T. (B) In vitro cytolytic function of control (non-transduced (NT)) and CAR.MUC1 T cells assessed in a 5-hour 51Cr-release assay at effector:targets of 5:1 to 40:1 using MUC1 + targets (BT-20, MDAMB-231) and MUC1- target (293T). Data represent mean±SEM (n=5). (C) Schematic representation of the TR2.41BB construct and transgenic expression of TR2.41BB T cells detected using biotinylated TR2. (D) NT and TR2.41BB transduced T cells were cultured alone or in the presence of rTR2 or anti-CD3 and anti-CD28. Cells were harvested at 120 min, the nuclear fraction was harvested, and an ELISA was performed to measure the translocation of NFκB into the nucleus. Data represent mean±SEM (n=3). Statistics: unpaired two-tailed t-test (B), two-way analysis of variance followed by Tukey’s multiple comparisons (D); *p<0.05; **p<0.01.

Article Snippet: 1 , Anti-MUC1 (Novus Biologicals) , SM3 , AF700.

Techniques: Expressing, In Vitro, Control, Release Assay, Construct, Transgenic Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Translocation Assay, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

doi: 10.1136/jitc-2021-003237

Figure Lengend Snippet: The TR2.41BB construct is able to rescue the cytotoxic activity of CAR.MUC1 in the presence of myeloid-derived suppressor cells (MDSCs). (A) Transgenic expression of CAR.MUC1.TR2.41BB T cells detected using anti-IgG2-CH3 spacer and biotinylated TR2, respectively, and quantitation of CAR.MUC1.TR2.41BB on T cells. Data represent mean±SEM (n=3). (B) CAR.MUC1 T cells were cultured with BT-20 (MUC1+) cells in the presence or absence of MDSCs and cytotoxic function was assessed in a 5-hour 51 Cr-release assay. Data represent mean±SEM (n=7). (C) CAR.MUC1, TR2.41BB, and CAR.MUC1.TR2.41BB T cells were cultured with BT-20 and 51 Cr-labeled MDSCs and the percentage of MDSC lysis was assessed in a 5-hour 51 Cr-release assay. Data represent mean±SEM (n=5). (D) CAR.MUC1, TR2.41BB, and CAR.MUC1.TR2.41BB T cells were cultured with 51 Cr-labeled BT-20 targets in the presence of MDSCs and cytotoxic function was assessed in a 5-hour 51 Cr-release assay. Data represent mean±SEM (n=5). Statistics: two-way analysis of variance followed by Tukey’s multiple comparisons (B, C, and D); *p<0.05; **p<0.01.

Article Snippet: 1 , Anti-MUC1 (Novus Biologicals) , SM3 , AF700.

Techniques: Construct, Activity Assay, Derivative Assay, Transgenic Assay, Expressing, Quantitation Assay, Cell Culture, Release Assay, Labeling, Lysis

Journal: Journal for Immunotherapy of Cancer

Article Title: Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer

doi: 10.1136/jitc-2021-003237

Figure Lengend Snippet: Expression of TR2.41BB enhances CAR.MUC1 T cell expansion, persistence, and antitumor activity leading to decreased metastasis in vivo in the presence of myeloid-derived suppressor cells (MDSCs). (A) Schematic of in vivo experiment in which NSG (NOD.CgPrkdcscid Il2rgtm1Wjl/SzJ) mice were transplanted with MDA-MB-231 cells with or without MDSCs and then treated with GFP.ffLuc-labeled NT, CAR.MUC1, TR2.41BB, or CAR.MUC1.TR2.41BB T cells. (B) Bioluminescence imaging indicating expansion and persistence of T cells and quantification of (C) bioluminescence at the tumor site and (D) tumor volume measured by calipers. Mean±SEM, n=4/group. (E) Schematic of in vivo experiment in which NSG mice were transplanted with GFP.ffLuc-labeled SUM-159 cells with or without MDSCs and treated with NT, CAR.MUC1, TR2.41BB, or CAR.MUC1.TR2.41BB T cells. (F) Tumor volume measured by calipers. Mean±SEM, n=5/group. Statistics: two-way analysis of variance followed by Tukey’s multiple comparisons (C, D, and F); *p<0.05; **p<0.01; ***p<0.001. CAR, chimeric antigen receptor; MUC1, Mucin 1; NT, non-transduced.

Article Snippet: 1 , Anti-MUC1 (Novus Biologicals) , SM3 , AF700.

Techniques: Expressing, Activity Assay, In Vivo, Derivative Assay, Labeling, Imaging

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Fluorescence analysis (relative fluorescence units, RFU) of endothelial cell ICAM-1, VCAM-1, E-selectin and P-selectin surface expression. For maximal stimulation, HUVEC were incubated with IL-1 (100 U/ml) for 12 h to up-regulate ICAM-1, and to induce VCAM-1 and E-selectin. HUVEC were incubated with PGE2 (10−6m) for 10 min to induce P-selectin expression. FL-1H (log) channel histogram analysis; 1 × 104 cells/scan. Control values were set at 100%. Mean ± s.d. of three experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Fluorescence, Expressing, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: MMF suppresses IL-1 induced E-selectin mRNA expression. Total RNA was isolated and E-selectin mRNA levels were determined by Northern blot analysis. GAPDH was used to assess equivalent RNA loading C−= unstimulated control cells, C+= IL-1 stimulated control cells. Results of densitometry are shown in the graph below and are expressed as the ratio of E-selectin : GAPDH mRNA, relative to the control (IL-1 activation without MMF), which was assigned a value of 100%. The figure shows one representative experiment from three.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Expressing, Isolation, Northern Blot, Control, Activation Assay

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Western blot analysis of P-selectin in endothelial cells. HUVEC were stimulated with 10−6m PGE2 and incubated additionally with different concentrations of MMF. Throm = control analysis of platelet P-selectin protein, C-= unstimulated HUVEC without MMF, C+ = PGE2-stimulated HUVEC without MMF. The figure shows one of four separate experiments.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Western Blot, Incubation, Control

Journal:

Article Title: The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

doi: 10.1046/j.1365-2249.2003.02290.x

Figure Lengend Snippet: Adhesion of CD4+ T (a), CD8+ T (b) or WiDr (c) cells to immobilized adhesion receptor globulin chimeras. Purified T cells or WiDr cells were added to culture dishes coated with the extracellular domain of ICAM-1, VCAM-1, E-selectin or P-selectin. The immobilized adhesion proteins were coupled to goat-antihuman IgG. Cells were pretreated with different concentrations of MMF. Untreated cells served as controls (100% value). Adhesion was measured after 30 min incubation and after removal of non-adherent cells, according to the protocol given in Materials and methods. Each point represents the mean ± s.d. of four experiments; s.d. was usually about 20%.

Article Snippet: After blocking, they were incubated overnight with sheep-antihuman P-selectin-antibodies (sheep IgG antibodies; 1 : 500 dilution; R&D Systems).

Techniques: Purification, Incubation