Journal: The Journal of Experimental Medicine

Article Title: Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions

doi: 10.1084/jem.20191157

Figure Lengend Snippet: Dynamic regulation of iNKT cell metabolism during development and the Mst1 dependence. (A and B) Basal OCR (A) and ECAR (B) in WT thymic CD4 + CD8 + (DP), S1–S2 iNKT (combined), and S3 iNKT cells. (C) OCR in WT DP, S1–S2 iNKT, and S3 iNKT cells after the addition of oligomycin (Oligo), FCCP, and rotenone (Rot). (D) SRC (= maximum OCR – baseline OCR) in WT S1–S2 iNKT and S3 iNKT cells. (E) Basal OCR/ECAR ratio in WT DP, S1–S2 iNKT, and S3 iNKT cells. (F) Confocal microscopy analysis of Tom20 (red, staining mitochondria) and actin (green) in WT DP, S1–S2 iNKT, and S3 iNKT cells (left). Scale bar, 5 µm. Right, quantification of mitochondrial volume per cell (μm 3 ; n = 10 cells/group). (G) Quantification of mitochondrial diameter (μm) using STORM imaging of WT DP, S1–S2 iNKT, and S3 iNKT cells. (H) Enriched pathways identified by GSEA from GO and HALLMARK gene sets in Mst1-deficient S3 iNKT cells compared with WT S3 iNKT cells. (I) Basal OCR and ECAR in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells ( n = 3 wells/group combined from 15 mice/group). (J) MFI of MitoTracker (left) and TMRM (right) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 3 or 4 mice/group). Histogram plots represent flow cytometry analysis of MitoTracker and TMRM in thymic S3 iNKT cells. (K) Confocal microscopy analysis (left) of Tom20 (red, staining mitochondria) and actin (green) in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells and quantification (right) of mitochondrial volume per cell (μm 3 ; n = 2 mice/group; n = 269–461 total cells from 10 fields). Scale bar, 5 µm. (L) STORM imaging (left) and quantification (right) of mitochondrial diameter (μm) in thymic WT and Cd4 Cre Mst1 fl/fl S3 iNKT cells ( n = 2 mice/group; n = 35–37 cells imaged/group). Scale bar, 1 µm. (M) Flow cytometry analysis (left) and MFI (right) of CellROX (cellular ROS) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 4 or 5 mice/group). (N) Flow cytometry analysis of phosphorylated S6 (pS6) in thymic S1–S2 and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice stimulated with or without PMA/ionomycin (PMA/iono) for 30 min ( n = 4–6 mice/group). Data are relative to the average pS6 MFI for WT cells. (O) MFI of ex vivo 2-NBDG uptake in S1, S2, and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice ( n = 5 mice/group). Numbers in the plots indicate MFI (J, M, and N). Data in plots indicate mean ± SEM (A–G and I–O). *, P < 0.05, **, P < 0.01, ***, P < 0.001. One-way ANOVA with Tukey’s test (A, B, and E–G), two-way ANOVA with Bonferroni’s test (C), or two-tailed unpaired Student’s t test (D, I, and J–O). Data are representative of two independent experiments (A–E, I, K, and L), or combined from two (F, G, and L) or three (J and M–O) independent experiments.

Article Snippet: For staining mitochondria, intracellular ROS, and 2-NBDG uptake, unfixed lymphocytes were incubated for 30 min at 37°C with 10 nM MitoTracker Green or MitoTracker Deep Red (Life Technologies, M7514 and MM22426), 10 nM TMRM (ImmunoChemistry Technologies, 9105), or 2.5 μM CellROX Deep Red (Life Technologies, C10422), or for 1 h at 37°C with 30 μM 2-NBDG (Thermo Fisher Scientific, N13195) before staining surface markers.

Techniques: Confocal Microscopy, Staining, Imaging, Flow Cytometry, Ex Vivo, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Hippo/Mst signaling coordinates cellular quiescence with terminal maturation in iNKT cell development and fate decisions

doi: 10.1084/jem.20191157

Figure Lengend Snippet: Opa1 enforces S3 iNKT and iNKT1 cell development and quiescence-associated features. (A) Immunoblot analysis of Opa1, VDAC (mitochondrial loading control), and β-actin (total protein loading control) in thymic S1–S2 and S3 iNKT cells from WT and Cd4 Cre Mst1 fl/fl mice. Opa1 expression was quantified relatively to VDAC expression ( n = 3–6 samples/group). (B and C) Flow cytometry analysis (upper) and percentages (lower) of total thymic iNKT cells and developmental stages (S1, S2, and S3; B) and effector cell subsets (C) in the thymus of WT and Cd4 Cre Opa1 fl/fl mice. (D) Flow cytometry analysis (left) and quantification of relative MFI (right) of MitoTracker (upper) or TMRM (middle) and percentages of Ki67 + cells (lower) in thymic S1, S2, and S3 iNKT cells from WT and Cd4 Cre Opa1 fl/fl mice ( n = 5 mice/group). MitoTracker and TMRM expression is relative to average MFI of WT S1 iNKT cells. (E) Confocal microscopy analysis (upper) of Tom20 (red, staining mitochondria) and actin (green) in S1–S2 and S3 iNKT cells from the thymus of WT and Cd4 Cre Opa1 fl/fl mice and quantification (lower) of mitochondrial volume per cell (μm 3 ; n = 2 mice/group; n = 20–91 total cells from three fields). Numbers indicate MFI or percentage of cells in gates or quadrants (B–D). Data in plots indicate mean ± SEM (A–E). *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. Two-tailed unpaired Student’s t test (A–E). Data are combined from three (A) or four (B–D) or representative of two (E) independent experiments.

Article Snippet: For staining mitochondria, intracellular ROS, and 2-NBDG uptake, unfixed lymphocytes were incubated for 30 min at 37°C with 10 nM MitoTracker Green or MitoTracker Deep Red (Life Technologies, M7514 and MM22426), 10 nM TMRM (ImmunoChemistry Technologies, 9105), or 2.5 μM CellROX Deep Red (Life Technologies, C10422), or for 1 h at 37°C with 30 μM 2-NBDG (Thermo Fisher Scientific, N13195) before staining surface markers.

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

Journal: Journal of Biological Chemistry

Article Title: α-Thrombin Rapidly Induces Tyrosine Phosphorylation of a Novel, 74–78-kDa Stress Response Protein(s) in Lung Fibroblast Cells

doi: 10.1074/jbc.m409043200

Figure Lengend Snippet: FIG. 4. The p74–78-kDa protein is localized to the cytoplasm. A, serum-starved cells were untreated or treated -thrombin (0.4 units/ml; 15 min) and lysed, and then membrane, cytoplasm, and nuclear frac- tions were prepared as described under “Experimental Procedures.” 15 g of the sample were run on an 8% SDS-polyacrylamide gel, trans- ferred to nitrocellulose, and immunoblotted with the phospho-specific anti-Stat3 antibody. The positions of three distinct bands (74–78 kDa) are indicated by arrows. B and C are blots probed with anti-Stat3 antibody and anti-TGF- receptor antibody, respectively. These blots are representative of three independent experiments. Th, -thrombin; CR, cross-reactive.

Article Snippet: IL-6, PDGF, transforming growth factor- (TGF- ), and basic fibroblast growth factor (FGF) were obtained from R & D Systems; nitrocellulose membrane was from Amersham Biosciences; phospho-specific antiStat3 antibody was from BIOSOURCE; anti-Stat3 antibody and antiE2F-1 antibody were from Santa Cruz Biotechnology; anti-phosphotyrosine antibody was from Upstate Biotechnology, Inc.; anti-GRP antibody was from Oxford Biomedical Research; goat anti-rabbit IgG and rabbit anti-mouse IgG were from Bio-Rad; all other chemicals were either from Sigma or Fisher.

Techniques: Membrane