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

Article Title: i NKT cells require autophagy to coordinate proliferation and survival signals during differentiation

doi: 10.4049/jimmunol.1402154

Figure Lengend Snippet: Loss of Atg5 expression led to cell cycle arrest. (A) Atg5 deficient iNKT cells exhibited enhanced incorporation of BrdU. 5 h after injection, thymic iNKT cells were collected and analyzed for BrdU incorporation and Ki-67 expression. (B) Thymic iNKT cells from Atg5f/f CD4-Cre mice accumulated in S-phase. 2 h post injection of BrdU, thymic iNKT cells were stained with 7-AAD and anti-BrdU antibody to evaluate cell cycle progression. (C) Increased expression of p21cip1 in Atg5 deficient iNKT cells. Shown are the representative flow cytometry analysis (left) and geometric MFI (right) of data pooled from at least three independent experiments gating on either NK1.1 or NK1.1+, CD1d tetramer+ thymocytes. Filled histogram: isotype control; solid line: wild type mice; dotted line: Atg5f/f CD4-Cre mice. * p<0.01, n≥4. Error bars are SD.

Article Snippet: Antibodies and reagents The following antibodies, with clone designation in parentheses, were from BD PharMingen: CD1d–PE (1B1), CD4-APC (RM4–5), CD8-PerCP-Cy5.5 (53–6.7), CD24-FITC (M1/69), CD45.1-FITC (A20), Fas-FITC (Jo2), anti-BrdU-Alexa Fluor 488 (3D4), IFNγ-PE-Cy7 (XMG1.2), IL-4-Alexa Fluor 647 (11B11), Ki-67-PE (B56), NK1.1-PE-Cy7 (PK136), GATA3-PE-Cy7 (L50-823), phospho-Akt(pS473)-PE (M89-61), phospho-Akt(pT308)-PE (J1–223.371) and purified antibody anti-active caspase 3 (C92-605).

Techniques: Expressing, Injection, BrdU Incorporation Assay, Staining, Flow Cytometry

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

Article Title: i NKT cells require autophagy to coordinate proliferation and survival signals during differentiation

doi: 10.4049/jimmunol.1402154

Figure Lengend Snippet: Atg5 deletion resulted in increased mitochondria, super oxide and cell death. Augmented mitochondrial mass (A) and mitochondrial superoxide production (B) in Atg5 deleted, thymic, NK1.1 and NK1.1+iNKT cells. (C) Higher expression of Fas on Atg5 deficient iNKT cells compared to wild type controls. At least four independent experiments were performed with consistent results, both representative flow cytometry plots (left) and geometric MFI values (right) are shown. Filled histogram: isotype control; solid line: wild type mice; dotted line: Atg5f/f CD4-Cre mice. (D) Dramatically increased apoptosis leading to cell death for NK1.1+ thymic iNKT cells devoid of Atg5. Cells were analyzed after overnight culture and compared with wild type controls. Representative flow cytometry analyses from three separate experiments (left) and geometric MFI (right) of dead cells (Annexin V+, Live/Dead Yellow+) are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n≥4. Error bars are SD.

Article Snippet: Antibodies and reagents The following antibodies, with clone designation in parentheses, were from BD PharMingen: CD1d–PE (1B1), CD4-APC (RM4–5), CD8-PerCP-Cy5.5 (53–6.7), CD24-FITC (M1/69), CD45.1-FITC (A20), Fas-FITC (Jo2), anti-BrdU-Alexa Fluor 488 (3D4), IFNγ-PE-Cy7 (XMG1.2), IL-4-Alexa Fluor 647 (11B11), Ki-67-PE (B56), NK1.1-PE-Cy7 (PK136), GATA3-PE-Cy7 (L50-823), phospho-Akt(pS473)-PE (M89-61), phospho-Akt(pT308)-PE (J1–223.371) and purified antibody anti-active caspase 3 (C92-605).

Techniques: Expressing, Flow Cytometry