limk inhibitor bms-5  (Bristol Myers)

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

Article Title: Human Natural Killer Cell Cytoskeletal Dynamics and Cytotoxicity are Regulated by LIM Kinase

doi: 10.4049/jimmunol.2000186

Figure Lengend Snippet: NK cells were exposed to the LIMK inhibitor BMS-5 for 48 hours prior to co-incubation with K562 target cells for 2 hours. (A) Confocal microscopy was utilized to image NK:target conjugates for intracellular F-actin (phalloidin AF555, red), perforin (AF488, green), and α-tubulin (AF647, blue) in control relative to 5 μM and 10 μM BMS-5 conditions. The NK cell is outlined for visualization. Quantitative analysis of F-actin accumulation at the immune synapse was measured by (B) area, (C) mean fluorescence intensity (MFI), and (D) integrated density (area x MFI) at the interface with the target cell. (E) The NK cell microtubule organizing center (MTOC) was identified as a discrete α-tubulin signal (as noted in panel A) and the distance from MTOC to the centroid of the immune synapse was calculated for each NK:target conjugate. Violin plots depict density distribution with median (line) and interquartile ranges (dashed line) with individual NK:target conjugate data points amongst LIMK inhibition conditions (white=10 μM BMS-5; light grey=5 μM BMS-5) relative to DMSO control (dark grey). (F) The relationship between F-actin density and MTOC to synapse distance was determined and Pearson correlation coefficient and significance are noted. (G) NK cell perforin density (area x MFI) at the synapse was quantified for each NK:target conjugate in n=3 experiments; 15–20 NK:target conjugates imaged per condition per experiment and experiments were pooled for analysis. Scale bar = 5 μm. **p<0.005, ***p<0.0005, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LIMK inhibition In select experiments, NK cells were exposed to the LIMK inhibitor BMS-5 (Enzo Life Sciences) at 1 μM, 5 μM, or 10 μM for 48 hours.

Techniques: Incubation, Confocal Microscopy, Fluorescence, Inhibition

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

Article Title: Human Natural Killer Cell Cytoskeletal Dynamics and Cytotoxicity are Regulated by LIM Kinase

doi: 10.4049/jimmunol.2000186

Figure Lengend Snippet: Human primary NK cells were exposed to increasing concentrations of the LIMK inhibitor BMS-5 for 48 hours in culture and protein was extracted. Protein expression was analyzed by Western immunoblotting for each of the indicated proteins and normalized to GAPDH as a loading control. Relative expression of (A) LIMK1, (B) LIMK2, (C) p-cofilin, and (D) cofilin was quantified after NK cells were exposed to 1 μM, 5 μM, and 10 μM BMS-5 and fold differences were calculated relative to vehicle control. (E) BMS-5-exposed NK cells were co-incubated with K562 target cells for 4 hours and target cell apoptosis was assessed by annexin V and 7-AAD staining as in the representative flow cytometry plots. Quadrant numbers indicate % of target cells. (F) Target cell apoptosis was quantified by annexin V staining and compared in LIMK inhibitor-exposed NK cells relative to control conditions for n=3–5 experiments. Bar graphs express mean ± SEM with individual data points. *p<0.05, **p<0.005 by one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: LIMK inhibition In select experiments, NK cells were exposed to the LIMK inhibitor BMS-5 (Enzo Life Sciences) at 1 μM, 5 μM, or 10 μM for 48 hours.

Techniques: Expressing, Western Blot, Incubation, Staining, Flow Cytometry

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

Article Title: Human Natural Killer Cell Cytoskeletal Dynamics and Cytotoxicity are Regulated by LIM Kinase

doi: 10.4049/jimmunol.2000186

Figure Lengend Snippet: NK cells were exposed to dexamethasone, LXA4, or vehicle control for 48 hours prior to co-incubation with K562 target cells for 2 hours. In some experiments, NK cells were pre-treated with the LIMK inhibitor BMS-5 (10 μM) prior to exposure to LXA4. (A) Confocal microscopy was utilized to image intracellular F-actin (phalloidin AF555, red), perforin (AF488, green), and α-tubulin (AF647, blue) in NK:target conjugates. (B) Immune synapse density of F-actin (area x MFI) was quantified in Veh- (grey), Dex- (blue), or LXA4-exposed (red) NK cells. (C) MTOC to immune synapse distance was calculated for individual NK:target conjugates in each condition. (D) The relationship between F-actin density and MTOC to synapse distance was determined and Pearson correlation coefficient and significance are noted. (E) Pie charts depict the percentage of NK cells with MTOC < 2 μm from the immune synapse (white) in each condition. The density of NK cell perforin within the immune synapse (F) and the entire NK cell (G) was calculated. ≥ 45 NK:target conjugates per condition were imaged from 3 individual experiments. NK:target conjugates were analyzed by confocal microscopy for (H) F-actin accumulation at the immune synapse, (I) MTOC to synapse distance, and (J) synaptic perforin content and (K) target cell apoptosis by flow cytometry in NK cells exposed to LXA4 in the presence or absence of the LIMK inhibitor BMS-5. (L) Expression of LIMK pathway proteins was assessed by Western immunoblotting of NK cells exposed to LXA4 in the presence or absence of the LIMK inhibitor BMS-5. n=2 experiments, ≥ 35 NK:target conjugates were imaged per condition. Violin plots show distribution density with median (line) and quartiles (dashed line). Bars express mean ± SEM. Scale bar = 5 μm. *p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test or two-tailed Student’s t-test.

Article Snippet: LIMK inhibition In select experiments, NK cells were exposed to the LIMK inhibitor BMS-5 (Enzo Life Sciences) at 1 μM, 5 μM, or 10 μM for 48 hours.

Techniques: Incubation, Confocal Microscopy, Flow Cytometry, Expressing, Western Blot, Two Tailed Test