Journal: International Journal of Molecular Sciences

Article Title: Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models

doi: 10.3390/ijms21228676

Figure Lengend Snippet: Dynamics of DOCK8 expression pattern in SK-N-BE(2) and SH-SY5Y over time. ( A – D ) Representative images of DOCK8 expression at the time points studied (w: weeks) in soft and stiff scaffoldings. Top images correspond to the SK-N-BE(2) cell line cultivated ( A ) alone and ( B ) with SW10; the images on the bottom represent the SH-SY5Y cell line cultivated (C) alone and ( D ) with SW10 cells. Scale bar 50 µm at top left of the first image. Same scale bar is valid for all images. ( E – H ) Bar chart quantification of DOCK8 staining (% of positive cells) for SK-N-BE(2) cells ( E ) alone and ( F ) SK-N-BE(2) cells plus SW10 cells in soft and stiff scaffolds, and SH-SY5Y cells ( G ) alone and ( H ) S-SY5Y cells plus SW10 cells in soft and stiff scaffolds. White and black bars: soft and stiff scaffolds, respectively. Dashed lines indicate moving average per stiffness condition. X axis: time in weeks (w) and Y axis: % of positive DOCK8 cells.

Article Snippet: ICC stains were performed with anti-Ki67 (prediluted, Agilent Technologies, Santa Clara, CA, USA), anti-VN (1/100, Abcam, Cambridge, UK), anti-DOCK8 (1/700, Invitrogen, Life Technologies, Waltham, MA, USA), anti-KANK1 (1/1000, Invitrogen, Life Technologies, USA), and anti-SYP (prediluted, Dako, Glostrup, Denmark) antibodies.

Techniques: Expressing, Staining

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A and B) Proliferation measured by Cell Trace Violet dilution (A) and IL-2 secretion in culture supernatants (B) by CD4+CD25– Teffs isolated from the spleens of Dock8–/– and WT mice cultured for 3 days with anti-CD3+anti-CD28–coated beads. (C) Percentage of CD25+FOXP3+ Tregs among CD4+ cells in the thymuses, spleens, and LNs of Dock8–/– and WT mice. n = 17 mice from each group for the thymus, n = 31 mice from each group for the spleen, n = 7 mice from each group for the LN. (D) Percentages of CD44–CD62Lhi rTregs and CD44+CD62Llo aTregs of total CD4+FOXP3+ cells in the spleens of Dock8–/– and WT mice. n = 5 mice from each group. (E) Representative FACS plots of intracellular FOXP3 and CTLA-4 and surface CD25 expression gating on CD4+FOXP3+ splenocytes (left). Quantitative analysis of surface CD25 expression by splenic CD4+FOXP3+ cells from Dock8–/– mice and WT controls. n = 29 mice from each group. (F) qPCR analysis of Foxp3 and Il2ra mRNA levels in FACS-sorted CD4+CD25+CD39+ Tregs from Dock8–/– and WT mice. Results are expressed as fold increase relative to the WT control ratio of the mRNA of interest/b2microglobulin. (G) Suppression of the proliferation of CD4+CD25– Teffs by CD4+CD25+CD39+ Tregs from Dock8–/– mice and WT controls. Teff proliferation was measured by FACS analysis of Cell Trace Violet dilution. The left panel is a representative experiment; the right panel shows the pooled results. (H) qPCR analysis of Tgfb and Il10 mRNA expression by sorted CD4+CD25+CD39+ Tregs from Dock8–/– mice and WT controls. Symbols represent individual mice, and error bars represent mean and SEM. Results in A, B, and F–H are representative of 3 independent experiments. t test, NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immune complexes were captured with protein G-Sepharose (EMD Millipore), washed, denatured by boiling in sample buffer, separated on acrylamide gels, and analyzed by immunoblotting with anti-DOCK8 (H-159, Santa Cruz) and anti-STAT5 (C-17, Santa Cruz).

Techniques: Isolation, Cell Culture, Expressing

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A) Representative FACS analysis of pSTAT5 staining (left) and percentage of pSTAT5+ cells (right) in splenic CD4+YFP+ Tregs from Foxp3YFP–Cre/Dock8flox/flox mice and Foxp3YFP–Cre controls stimulated for 15 minutes with 10 ng/ml mouse IL-2. (B) Relative pSTAT5 content in CD4+FOXP3+YFP– and CD4+FOXP3+YFP+ Tregs from Foxp3YFP–Cre/+/Dock8flox/flox female mice and Foxp3YFP–Cre/+ controls at baseline and following stimulation with increasing concentrations of IL-2 for 15 minutes. The graph plots the relative pSTAT5/STAT5 MFI ratio normalized to the Foxp3YFP–Cre/+ YFP– Treg baseline ratio. (C) Representative immunoblot of STAT5, pSTAT5, and DOCK8 in DOCK8 immunoprecipitates and cell lysates from WT T cells, and quantification of 4 individual experiments, showing the ratio of immunoprecipitated STAT5/DOCK8. Results were normalized to media alone samples. (D) Representative immunoblot of STAT5, pSTAT5, and DOCK8 in STAT5 immunoprecipitates and cell lysates from WT T cells, and quantification of 3 individual experiments, showing the ratio of immunoprecipitated DOCK8/STAT5. Results were normalized to media-alone samples. Results in A and B are representative of 3 independent experiments using 2–3 mice per group in each. Error bars represent the mean and SEM. Significance was determined by unpaired t test in A, C, and D, while ANOVA was used to compare the curves in B. ns P > 0.05, ***P < 0.001.

Article Snippet: Immune complexes were captured with protein G-Sepharose (EMD Millipore), washed, denatured by boiling in sample buffer, separated on acrylamide gels, and analyzed by immunoblotting with anti-DOCK8 (H-159, Santa Cruz) and anti-STAT5 (C-17, Santa Cruz).

Techniques: Staining, Western Blot, Immunoprecipitation

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A) Baseline F-actin content of CD4+CD25+ Tregs from Dock8–/– and WT mice, and effect of CD3 crosslinking on the F-actin content of Tregs from Dock8–/– and WT mice. Results are expressed as the change in the MFI of F-actin from the baseline (time 0). (B) Representative images of CD4+CD25+ Tregs from Dock8–/– mice and WT controls plated on anti-CD3– and ICAM-1–coated glass chambers and stained for F-actin by phalloidin at 5 and 45 minutes (original magnification, ×100). (C) Relative number of adherent cells per unit area at 5 minutes. (D) Quantitative analysis of F-actin staining of Tregs after 5 and 45 minutes of stimulation. (E) Localization of pTYR, F-actin, and DOCK8 in Dock8–/– and WT Tregs at 10 minutes (original magnification, ×100). (F) A representative TIRF image of DOCK8 and actin distribution across the synapse after 10 minutes of synapse formation. The graph shows line scan profiles of DOCK8 and ACTIN across the middle of the cell; the green trace represents ACTIN intensity distribution, and the red trace represents DOCK8 intensity distribution. Note that DOCK8 and ACTIN intensities coincide in the cell periphery, represented by the peaks at the beginning and end of the line scan profiles. (G) Side view of 2 representative T cells, showing the whole cell ACTIN, pTYR, and DOCK8 distribution. Cells stained with DOCK8, pTYR, and ACTIN were imaged using confocal microscopy, and the images show a side view of maximum intensity projection of the confocal images. (H) Quantification of cell edge roughness determined by “shape descriptors” utility of ImageJ at 5 and 45 minutes. (I) Measurement of immune synapse instability, as denoted by the kinapse index (transient interactions index > 1) at 5 and 45 minutes. (J) Relative staining intensity of pTYR, phospho-ZAP70 (pZAP70), and TALIN at 10 minutes. (K) Transendocytosis of CD86-GFP by Tregs from Dock8–/– and WT mice cocultured with CD86-GFP–expressing CHO cells. The percentage of GFP+ Tregs of total Tregs is shown. Results in A and K are representative of 3 independent experiments. Results in B–J are representative of 2 independent experiments. Symbols represent individual measurements, and error bars represent mean and SEM. Significance was determined by unpaired t test. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Immune complexes were captured with protein G-Sepharose (EMD Millipore), washed, denatured by boiling in sample buffer, separated on acrylamide gels, and analyzed by immunoblotting with anti-DOCK8 (H-159, Santa Cruz) and anti-STAT5 (C-17, Santa Cruz).

Techniques: Staining, Confocal Microscopy, Expressing

Journal: JCI Insight

Article Title: DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs

doi: 10.1172/jci.insight.94298

Figure Lengend Snippet: (A and B) CD4+CD25+CD39+YFP+ (DOCK8-deficient) and YFP– (DOCK8-sufficient) Tregs were sorted from Foxp3YFP–Cre/+/Dock8flox/flox female mice. Tregs were cultured overnight in media alone (A) or with anti-CD3+CD28 beads (B). Heatmaps of selected genes differentially expressed in YFP+ and YFP– Tregs from Foxp3YFP–Cre/+Dock8flox/flox female mice. The cutoff for significance was P < 0.05. P values were calculated using the Wald test for differential expression analysis. P values were corrected afterward for multiple testing. Expression of genes is centered and scaled by row to highlight differences in each gene sample. Each column represents an individual mouse. (C and D) CD4+CD25+CD39+ Tregs were FACS sorted from Dock8–/– and WT mice. RNA was prepared from Tregs directly after isolation (C) or after 24-hour culture with anti-CD3+CD28 beads (C and D). qPCR results are expressed as fold increase of mRNA of interest/b2microglobulin ratio relative to the unstimulated WT Tregs. (E) MFI of surface marker staining on YFP– DOCK8-sufficient and YFP+ DOCK8-deficient CD4+CD25+CD39+ Tregs from Foxp3YFP–Cre/+/Dock8flox/flox female mice. Symbols represent individual mice. Bars in C–E represent the mean and SEM. t test, NS P > 0.05, *P < 0.05, ***P < 0.001.

Article Snippet: Immune complexes were captured with protein G-Sepharose (EMD Millipore), washed, denatured by boiling in sample buffer, separated on acrylamide gels, and analyzed by immunoblotting with anti-DOCK8 (H-159, Santa Cruz) and anti-STAT5 (C-17, Santa Cruz).

Techniques: Cell Culture, Expressing, Isolation, Marker, Staining