Journal: The FASEB Journal

Article Title: Activation of JAK/STAT3 restores NK-cell function and improves immune defense after brain ischemia

doi: 10.1096/fj.201700962R

Figure Lengend Snippet: Gene overlap between poststroke NK-cell gene profile and NK suppression–induced infection gene network

Article Snippet: NK cells were sorted from the pooled splenocytes of wild-type mice and transfected with STAT3 CRISPR activation plasmid or CRISPR-control plasmid (sc-437275; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Techniques: Infection, Virus

Journal: The FASEB Journal

Article Title: Activation of JAK/STAT3 restores NK-cell function and improves immune defense after brain ischemia

doi: 10.1096/fj.201700962R

Figure Lengend Snippet: Genetic activation of STAT3 preserved NK-cell–derived IFN-γ expression after MCAO. C57BL/6 mice were subjected to sham operation or MCAO surgery. A, B) Real-time PCR and flow cytometry plots showed STAT3 gene (A) and protein levels (B) in splenic NK cells 24 h after MCAO; n = 4 per group. C) The schematic graph of the experimental design. Splenic NK cells isolated from wild-type mice were treated with STAT3-CRISPR activation plasmid or control vector. After transfected for 48 h, 3 × 106 NK cells were transferred intraveniously into groups of Rag2−/−γc−/− mice before MCAO. D) Flow cytometry plots and bar graph showed STAT3 phosphorylation in NK cells 2 h after MCAO; n = 4/group. E, F) Flow cytometry plots (E) and bar graph (F) show the effect of STAT3 activation on functional marker expression (CD69, perforin, and IFN-γ) of splenic NK cells at d 1 after MCAO. Plots represent the results from 3 independent experiments. Error bars represent mean ± sem. **P < 0.01 by 2-tailed, unpaired Student’s t test.

Article Snippet: NK cells were sorted from the pooled splenocytes of wild-type mice and transfected with STAT3 CRISPR activation plasmid or CRISPR-control plasmid (sc-437275; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Isolation, CRISPR, Plasmid Preparation, Control, Transfection, Phospho-proteomics, Functional Assay, Marker

Journal: The FASEB Journal

Article Title: Activation of JAK/STAT3 restores NK-cell function and improves immune defense after brain ischemia

doi: 10.1096/fj.201700962R

Figure Lengend Snippet: STAT3 activation in NK cells improves survival and reduces lung bacterial burden in MCAO mice. Splenic NK cells isolated from C57BL/6 mice targeting STAT3 were established by introducing a STAT3 activation plasmid. STAT3-activated (STAT3-CRISPR) NK cells were then passively transferred via intravenious injection to Rag2−/−γc−/− transgenic mice, followed by MCAO surgery. A) Representative MRI images and summarized data showed stroke lesion volume after adoptive transfer with STAT3-CRISPR NK cells; n = 5 mice/group. B) The neurodeficit score was assessed by modified neurological severity score (mNSS) after STAT3-CRISPR NK-cell transfer and MCAO surgery. P > 0.05 by 2-way ANOVA (A, B). C) The survival of the mice was noted at 0 to 8 d after cell transfer and MCAO surgery; n = 15 mice/group. *P < 0.05 by 2-way ANOVA. D) Lung tissues from MCAO mice transferred with STAT3-CRISPR NK cells were collected for bacteriologic analysis at 3 d after MCAO surgery. Data summarized in D graphically illustrate the quantification of bacteria burden in lung after passive transfer with NK cells. Data are presented in colony-forming units (CFU) per organ lung tissue homogenate. **P < 0.01 by 2-tailed, unpaired Student’s t test. E) Hematoxylin and eosin–stained lung sections exhibited typical signs (thickening of alveolar walls and neutrophilic infiltrates) of bacterial burden in MCAO mice. The lung tissues from wild-type mice adoptively transferred with control vector transfected-NK or STAT3-CRISPR NK cells were collected at d 3 after MCAO for histologic examination. Scale bars, 50 μm. F) ELISA measurement of IFN-γ protein levels in serum from the control or STAT3-CRISPR NK groups at d 3 after MCAO. **P < 0.01 by 2-tailed, unpaired Student’s t test. Mean ± sem.

Article Snippet: NK cells were sorted from the pooled splenocytes of wild-type mice and transfected with STAT3 CRISPR activation plasmid or CRISPR-control plasmid (sc-437275; Santa Cruz Biotechnology) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Isolation, Plasmid Preparation, CRISPR, Injection, Transgenic Assay, Adoptive Transfer Assay, Modification, Bacteria, Staining, Control, Transfection, Enzyme-linked Immunosorbent Assay