Journal: The Journal of Immunology Author Choice

Article Title: RNA helicase DDX3X promotes NK cell survival by supporting MCL1 expression

doi: 10.1093/jimmun/vkaf281

Figure Lengend Snippet: RNA helicase DDX3 supports the survival of mouse NK cells. Male mouse NK cells were isolated from the spleens of (A) C57BL/6 or (B) Cas9 transgenic mice using negative selection columns and incubated in culture medium supplemented with IL-2 and IL-15. (A) The DDX3 inhibitor RK33 (DDX3-i, red) or vehicle (DMSO, black) were added to cultures for 20 hours. Representative flow plots reveal uptake of NIR Zombie live/dead dye and loss of viability, with median and individual results for 5 mice/group. Statistical significance determined by Mann–Whitney test. Results are representative of >3 independent experiments. (B) Control or Ddx3x sgRNA (20 µM) was transfected into separate pools of Cas9 transgenic NK cells. After 48 hours, the viability of NK cells was assessed by flow cytometry, with representative and median outcomes displayed. Statistical significance determined by unpaired 2-tailed Student t -test. Data are representative of 2 independent experiments. (C) Immunoblot showing β-actin and DDX3X expression in isolated mouse NK cells from Cas9 transgenic mice 24 hours after transfection with scrambled or Ddx3x -specific sgRNA.

Article Snippet: C57BL/6 (strain #00064) and Cas9 transgenic (strain #024857) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Isolation, Transgenic Assay, Selection, Incubation, MANN-WHITNEY, Control, Transfection, Flow Cytometry, Western Blot, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: RNA helicase DDX3X promotes NK cell survival by supporting MCL1 expression

doi: 10.1093/jimmun/vkaf281

Figure Lengend Snippet: DDX3X is required to sustain expression of the prosurvival protein MCL1. Mouse NK cells were isolated from the spleens of male (A–D) C57BL/6 or (E) Cas9 mice and cultured with IL-2/IL-15. (A) Cultures were treated with RK-33 (DDX3-i, red) or DMSO (black) for 24 hours prior to flow cytometry–based assessment of expression of prosurvival proteins BCL2, BCL-xL, and MCL1. Histograms depict representative staining and dot plots show individual mouse mean fluorescent intensity (MFI) and group mean. (B–D) Cultured C57BL/6 NK cells were transfected with scrambled (control) or (B–C) Ddx3x -specific siRNA or (C–D) Ddx3y-specific siRNA for 24 hours before flow cytometry analysis of cell viability. (B, D) Histograms depict representative intensity of prosurvival protein expression while dot plots show individual mouse MFI and group mean. (C) Real-time quantification of Ddx3x and Ddx3y transcripts in siRNA-treated cells normalized to expression in controls. (D) Cultured NK cells from Cas9 transgenic mice were transfected with 20 µM Ddx3x or control sgRNA. After 48 hours, MCL1 expression levels were determined and are presented as described for (A). Statistical significance determined by Mann–Whitney test. Results are representative of at least 2 independent experiments.

Article Snippet: C57BL/6 (strain #00064) and Cas9 transgenic (strain #024857) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Expressing, Isolation, Cell Culture, Flow Cytometry, Staining, Transfection, Control, Transgenic Assay, MANN-WHITNEY

Journal: Biomedical Optics Express

Article Title: Histology correlated adaptive optics polarisation sensitive optical coherence tomography

doi: 10.1364/BOE.590238

Figure Lengend Snippet: Top row: log-scale reflectance and DOPU of albino mouse retina. Second row: log-scale reflectance and DOPU of C57BL/6 mouse retina. Third row: log-scale reflectance and DOPU of Cx3cr1 GFP Ccr2 RFP mouse retina. Retinal layers as indicated on reflectance image in top row: retinal nerve fibre layer (RNFL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); external limiting membrane (ELM); photoreceptor outer segments (OS); photoreceptor inner segments (IS); retinal pigment epithelium (RPE); choroid.

Article Snippet: Cx3cr1 GFP Ccr2 RFP transgenic mice (Jackson Laboratory, strain no. 032127; RRID: IMSR_JAX:032127) were used to generate time-course images.

Techniques: Membrane