Journal: Science Advances

Article Title: KRAS G12V /HLA-A*02:01–targeted chimeric antigen receptor T cells exhibit potent preclinical activity against solid tumors

doi: 10.1126/sciadv.aea2511

Figure Lengend Snippet: ( A ) Histomorphological validation of PDOs. Top: Hematoxylin and eosin (H&E) staining of primary tumor tissues from three independent patients with CRC (scale bars, 50 μm). Middle: Bright-field images of the matched PDOs (scale bars, 100 μm). Bottom: H&E staining of PDOs showing maintained tumor architecture (scale bars, 50 μm). ( B ) Caspase-3 (c-cas3)–dependent apoptosis in organoids after 24 hours of coculture with CAR T cells at a 1:1 E:T ratio was quantified by cleaved caspase-3 ELISA ( n = 3 biological replicates). ** P < 0.01. ( C ) Levels of TNF-α (left) and IFN-γ (right) released by CD19 CAR T cells and B9 CAR T cells in the culture supernatant of coculture with PDOs after 24 hours at a 1:1 E:T ratio ( n = 3 biological replicates). Data are represented as the means ± SD. Significance determined by two-way ANOVA with Tukey’s post hoc test: *** P < 0.001.

Article Snippet: Supernatants collected after 24 hours were analyzed for cytokines, while apoptosis was determined by cleaved caspase-3 ELISA (BOSTER, EK1425) per kit instructions.

Techniques: Biomarker Discovery, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Neuroprotective Effects and Mechanisms of Zhenlong Xingnao Capsule in In Vivo and In Vitro Models of Hypoxia

doi: 10.3389/fphar.2019.01096

Figure Lengend Snippet: Effects of ZXC on the mRNA levels of the Bcl-2/Bax ratio (A) , caspase-3 (B) , nuclear factor (NF)-кB (C) , and p38 (D) in the prefrontal cortex of ischemia-reperfusion injury rats. The data are expressed as mean ± standard deviation (n = 3). I-30, ischemia for 30 min; I-90, ischemia for 90 min; I-90+R-30, ischemia for 90 min, then reperfusion for 30 min; I-90+R-180, ischemia for 90 min, then reperfusion for 180 min. * P < 0.05 vs. sham group; # P < 0.05 vs. model group; ## P < 0.01 vs. model group.

Article Snippet: The primary antibodies used for IHC were the MAPK14 (p38) antibody and CASP3 (P17) antibody; both were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: Neuroprotective Effects and Mechanisms of Zhenlong Xingnao Capsule in In Vivo and In Vitro Models of Hypoxia

doi: 10.3389/fphar.2019.01096

Figure Lengend Snippet: Western blotting results for Sham, Model and ZXC groups (A) . Effects of ZXC on the protein expressions Bcl-2 (B) , Bax (C) , Caspase-3 (D) , NF-кB (E) , and p38 (F) in brain tissue of ischemia-reperfusion injury rats induced by MCAO. The data are expressed as mean ± standard deviation (n = 4). I-30, ischemia for 30 min; I-90, ischemia for 90 min; I-90+R-30, ischemia for 90 min, then reperfusion for 30 min. *P < 0.05 vs. sham group; # P < 0.05 vs. model group.

Article Snippet: The primary antibodies used for IHC were the MAPK14 (p38) antibody and CASP3 (P17) antibody; both were obtained from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Standard Deviation

Journal: The Journal of Neuroscience

Article Title: Neuronal Interleukin-16 (NIL-16): A Dual Function PDZ Domain Protein

doi: 10.1523/JNEUROSCI.19-18-07770.1999

Figure Lengend Snippet: Primary sequence of NIL-16. PDZ domains areunderlined; GLGF motifs (Cho et al., 1992) areboxed; amino acid numbers are indicated atleft and right. The horizontal arrow marks the start of splenocyte-derived pro-IL-16 (Keane et al., 1998). The vertical arrow indicates the site of proteolytic cleavage of pro-IL-16 by caspase-3 (Zhang et al., 1998).

Article Snippet: One aliquot per transcript received 0.5 μl (100 ng) purified, active recombinant human caspase-3 (PharMingen).

Techniques: Sequencing, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Neuronal Interleukin-16 (NIL-16): A Dual Function PDZ Domain Protein

doi: 10.1523/JNEUROSCI.19-18-07770.1999

Figure Lengend Snippet: Processing of NIL-16 by caspase-3.A, In vitro caspase-3 cleavage assay. Radiolabeled NIL-16 and pro-IL-16 were generated by in vitro translation and incubated in the presence or absence of recombinant, active caspase-3. Reaction products were separated by SDS-PAGE and analyzed by autoradiography. The positions of unprocessed NIL-16 and pro-IL-16 as well as that of the cleavage product IL-16 are indicated on the right. B, Processing of NIL-16 and pro-IL-16 in transfected COS-7 cells. COS-7 cells have an intrinsic caspase-3-like activity. Therefore, the processing of NIL-16 and pro-IL-16 was analyzed in these cells. Lysates of NIL-16-, pro-IL-16-, and mock-transfected cells as well as conditioned medium from the cultures were subjected to Western analysis. The primary antibody used in the experiment recognized the IL-16 epitope. Full-length proteins were detected in the respective cell lysates, whereas processed, mature IL-16 was found secreted into the medium of both NIL-16- and pro-IL-16-transfected cells. The positions of molecular weight markers in the gel are indicated on theright.

Article Snippet: One aliquot per transcript received 0.5 μl (100 ng) purified, active recombinant human caspase-3 (PharMingen).

Techniques: In Vitro, Cleavage Assay, Generated, Incubation, Recombinant, SDS Page, Autoradiography, Transfection, Activity Assay, Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: Neuronal Interleukin-16 (NIL-16): A Dual Function PDZ Domain Protein

doi: 10.1523/JNEUROSCI.19-18-07770.1999

Figure Lengend Snippet: Processing of NIL-16 and caspase-3 activation in cultured cerebellar granule neurons (CGN) after the induction of apoptosis. A, Apoptosis of CGN was induced by a medium switch to nondepolarizing conditions. Cell lysates were prepared at various times thereafter (indicated above the lanes) and subjected to Western analysis. The antibody used for the detection of NIL-16 recognized the IL-16 epitope, which is cleaved off the C terminus by caspase-3. The anti-caspase-3 antibody recognized both the inactive proenzyme (p32) and the p17 subunit of the active caspase. Caspase-3 activation was detected at all time points analyzed after the medium switch. In parallel, immunoreactivity against full-length NIL-16 was diminished progressively. B, The caspase inhibitor Z-VAD was included in the medium at the time of the switch to nondepolarizing conditions. Western analysis of cell lysates was performed as described in A. Under these conditions the caspase-3 activation and NIL-16 processing were greatly reduced. The positions of NIL-16, pro-caspase-3, and active caspase-3 (p17) in the gel are indicated on the left.

Article Snippet: One aliquot per transcript received 0.5 μl (100 ng) purified, active recombinant human caspase-3 (PharMingen).

Techniques: Activation Assay, Cell Culture, Western Blot

Journal: Journal of Immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for Pre-B Cell Loss in V H -Mutant Rabbits

doi: 10.4049/jimmunol.1101778

Figure Lengend Snippet: Flow cytometric analysis of ali/ali and wt B-lineage cell survival 2–6 d after transfer of pro-B cells to OP9 stroma in the absence of IL-7. A, Relative number of B-lineage (CD79a+) cells normalized to ali/ali cultures using bead-based counting and gating strategy in Fig. 2A. Error bars show SD from three independent experiments. B, Live/Dead staining of total lymphocytes. C, Cross-reactivity of anti–caspase-3 Ab with a UV-irradiated rabbit B cell line (55D1). Left panel, UV-treated 55D1 B cells immediately after irradiation (t0) (gray filled) or 3 h postirradiation stained with anti–caspase-3 (black dashed line) or isotype control for 3 h time point (solid black line) (identical result was observed for isotype control staining at t0); number is percentage of caspase-3+ cells at 3 h. Right panel, CD79a expression in caspase-3+ (dashed line) and caspase-3neg (solid line) cells 3 h postirradiation. D, Frequency of active caspase-3+ B-lineage cells (CD79a+). Data for B and D are representative of three independent experiments.

Article Snippet: Abs and flow cytometry Abs used: mouse anti-human CD79a-PE (catalog number 555935) or APC (catalog number 551134), rabbit anti-human caspase-3–FITC (catalog number 559341), mouse anti-human spleen tyrosine kinase (Syk)-P-Y348 (I1s0-722), mouse anti-rabbit–μ (367.2-bio), and rat anti-mouse pre-BCR (SL-156-bio) from BD Pharmingen (San Diego, CA); rat anti-mouse–μ-PE and PE-Cy7 (II/41) from eBioscience (San Diego, CA); goat F(ab′) 2 anti-human IgH+L, rabbit F(ab)-FITC anti-goat IgG H+L, and allophycocyanin-conjugated streptavidin from Jackson ImmunoResearch Laboratories (West Grove, PA); and biotinylated rabbit anti–a2-allotype and FITC-rabbit anti–a2-allotype IgG (generated by K.L.K.’s laboratory).

Techniques: Staining, Irradiation, Expressing