Journal: Autophagy

Article Title: Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

doi: 10.4161/auto.6.4.11811

Figure Lengend Snippet: IFNα induces STAT activation, stable transcription factor changes, and stable Th1/Tc1 polarization. Human T cells were stimulated for 6 days with anti-CD3 and anti-CD28 in media containing IL2 and IFNα either with rapamycin (T1.R) or without rapamycin (T1). (A) Control T1 and T1.R cells at day 6 were evaluated by IC flow for activation (phosphorylation) of STAT1, STAT4, STAT3 and STAT5 (% STAT+ cells; pooled results of n = 3 experiments). (B) Control T1 and T1.R cell expression of T-bet, GATA-3 and Foxp3 (% transcription factor+ cells; n = 3). (C) Day 6 T1.R cells were re-stimulated and maintained in the same culture conditions for an additional six days (first column), expanded in high-dose IL-2 (second column), expanded in high-dose IL-2 plus IL-4 (third column), expanded with lack of co-stimulation (fourth column), or expanded without rapamycin addition (fifth column). On day 12 of culture, the frequency of T cells expressing T-bet, GATA-3 and Foxp3 was determined (parts i–iii, respectively). (D) At day 12 of culture, T cells maintained in these five separate conditions were re-stimulated, and the resultant supernatants were evaluated for content of IFNγ (part i) and IL-4 (part ii).

Article Snippet: Flow cytometry to assess phosphorylation status of STAT molecules was performed using BD phosphoflow kit containing BD lyse/fix buffer and perm buffer III (558050); STAT4 PE (clone 38; p-stat4, 558249), STAT1 A488 (clone 4a; p-stat1, 560191), STAT3 A647 (clone49; p-stat3, 557815) and STAT5 PB (clone47: p-stat5, 560311) antibodies were utilized.

Techniques: Activation Assay, Expressing

Journal: Autophagy

Article Title: Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

doi: 10.4161/auto.6.4.11811

Figure Lengend Snippet: IFNα induces STAT activation, stable transcription factor changes, and stable Th1/Tc1 polarization. Human T cells were stimulated for 6 days with anti-CD3 and anti-CD28 in media containing IL2 and IFNα either with rapamycin (T1.R) or without rapamycin (T1). (A) Control T1 and T1.R cells at day 6 were evaluated by IC flow for activation (phosphorylation) of STAT1, STAT4, STAT3 and STAT5 (% STAT+ cells; pooled results of n = 3 experiments). (B) Control T1 and T1.R cell expression of T-bet, GATA-3 and Foxp3 (% transcription factor+ cells; n = 3). (C) Day 6 T1.R cells were re-stimulated and maintained in the same culture conditions for an additional six days (first column), expanded in high-dose IL-2 (second column), expanded in high-dose IL-2 plus IL-4 (third column), expanded with lack of co-stimulation (fourth column), or expanded without rapamycin addition (fifth column). On day 12 of culture, the frequency of T cells expressing T-bet, GATA-3 and Foxp3 was determined (parts i–iii, respectively). (D) At day 12 of culture, T cells maintained in these five separate conditions were re-stimulated, and the resultant supernatants were evaluated for content of IFNγ (part i) and IL-4 (part ii).

Article Snippet: Flow cytometry to assess phosphorylation status of STAT molecules was performed using BD phosphoflow kit containing BD lyse/fix buffer and perm buffer III (558050); STAT4 PE (clone 38; p-stat4, 558249), STAT1 A488 (clone 4a; p-stat1, 560191), STAT3 A647 (clone49; p-stat3, 557815) and STAT5 PB (clone47: p-stat5, 560311) antibodies were utilized.

Techniques: Activation Assay, Expressing

Journal: Autophagy

Article Title: Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

doi: 10.4161/auto.6.4.11811

Figure Lengend Snippet: Th1/Tc1 polarization during induction of rapamycin resistance requires PI3 kinase. (A) Human Th1/Tc1 cells were generated by co-stimulation and six-day culture in IL-2 + IFNα either alone (“−”) or in the presence of rapamycin (“+”). The protein lysate was obtained and subjected to western blot analysis for the following molecules: β-Actin, mTOR/phospho-mTOR, p70S6 kinase/phospho-p70S6 kinase, and 4EBP1. A similar result was obtained in two additional experiments. (B) Protein lysates from human Th1/Tc1 cells (“−”) or T1.R cells (“+”) were also tested for expression of total PI3 kinase (“PI3K”) or two forms of phosphorylated PI3 kinase (“p85 Tyr458” and “p85 Tyr 199”). A similar result was obtained in two additional experiments. (C) Representative examples of STAT1 phosphorylation in T1.R cells by flow cytometry analysis after restimulation alone (part i) or with the PI3 kinase inhibitor, LY294002 (part ii); summation of results using PI3K inhibition by LY in T1 cells (“LY”; part iii, n = 3), T1.R cells (part iv, n = 3) or by wortmannin (“W”; part v, n = 3). (D) Evaluation of PI3K dependency of type I polarization of T1 cells using LY (part ii, n = 3), T1.R cells using LY (part i; representative example of three experiments); results are expressed as frequency of CD4+ or CD8+ T cells that express IFNγ by IC flow cytometry after re-stimulation. Similar PI3K-dependency was observed using wortmannin as inhibitor (part iii; summation data of n = 3 experiments). (E) PI3K-dependency of T1.R polarization was also evaluated by testing supernatants for IL-2 and IFNγ content (representative example with LY as inhibitor, (parts i and ii); summation of n = 3 experiments using wortmannin as inhibitor, parts iii and iv).

Article Snippet: Flow cytometry to assess phosphorylation status of STAT molecules was performed using BD phosphoflow kit containing BD lyse/fix buffer and perm buffer III (558050); STAT4 PE (clone 38; p-stat4, 558249), STAT1 A488 (clone 4a; p-stat1, 560191), STAT3 A647 (clone49; p-stat3, 557815) and STAT5 PB (clone47: p-stat5, 560311) antibodies were utilized.

Techniques: Generated, Western Blot, Expressing, Flow Cytometry, Inhibition

Journal: Autophagy

Article Title: Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

doi: 10.4161/auto.6.4.11811

Figure Lengend Snippet: IFNα induces STAT activation, stable transcription factor changes, and stable Th1/Tc1 polarization. Human T cells were stimulated for 6 days with anti-CD3 and anti-CD28 in media containing IL2 and IFNα either with rapamycin (T1.R) or without rapamycin (T1). (A) Control T1 and T1.R cells at day 6 were evaluated by IC flow for activation (phosphorylation) of STAT1, STAT4, STAT3 and STAT5 (% STAT+ cells; pooled results of n = 3 experiments). (B) Control T1 and T1.R cell expression of T-bet, GATA-3 and Foxp3 (% transcription factor+ cells; n = 3). (C) Day 6 T1.R cells were re-stimulated and maintained in the same culture conditions for an additional six days (first column), expanded in high-dose IL-2 (second column), expanded in high-dose IL-2 plus IL-4 (third column), expanded with lack of co-stimulation (fourth column), or expanded without rapamycin addition (fifth column). On day 12 of culture, the frequency of T cells expressing T-bet, GATA-3 and Foxp3 was determined (parts i–iii, respectively). (D) At day 12 of culture, T cells maintained in these five separate conditions were re-stimulated, and the resultant supernatants were evaluated for content of IFNγ (part i) and IL-4 (part ii).

Article Snippet: Flow cytometry to assess phosphorylation status of STAT molecules was performed using BD phosphoflow kit containing BD lyse/fix buffer and perm buffer III (558050); STAT4 PE (clone 38; p-stat4, 558249), STAT1 A488 (clone 4a; p-stat1, 560191), STAT3 A647 (clone49; p-stat3, 557815) and STAT5 PB (clone47: p-stat5, 560311) antibodies were utilized.

Techniques: Activation Assay, Expressing

Journal: Autophagy

Article Title: Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

doi: 10.4161/auto.6.4.11811

Figure Lengend Snippet: IFNα induces STAT activation, stable transcription factor changes, and stable Th1/Tc1 polarization. Human T cells were stimulated for 6 days with anti-CD3 and anti-CD28 in media containing IL2 and IFNα either with rapamycin (T1.R) or without rapamycin (T1). (A) Control T1 and T1.R cells at day 6 were evaluated by IC flow for activation (phosphorylation) of STAT1, STAT4, STAT3 and STAT5 (% STAT+ cells; pooled results of n = 3 experiments). (B) Control T1 and T1.R cell expression of T-bet, GATA-3 and Foxp3 (% transcription factor+ cells; n = 3). (C) Day 6 T1.R cells were re-stimulated and maintained in the same culture conditions for an additional six days (first column), expanded in high-dose IL-2 (second column), expanded in high-dose IL-2 plus IL-4 (third column), expanded with lack of co-stimulation (fourth column), or expanded without rapamycin addition (fifth column). On day 12 of culture, the frequency of T cells expressing T-bet, GATA-3 and Foxp3 was determined (parts i–iii, respectively). (D) At day 12 of culture, T cells maintained in these five separate conditions were re-stimulated, and the resultant supernatants were evaluated for content of IFNγ (part i) and IL-4 (part ii).

Article Snippet: Flow cytometry to assess phosphorylation status of STAT molecules was performed using BD phosphoflow kit containing BD lyse/fix buffer and perm buffer III (558050); STAT4 PE (clone 38; p-stat4, 558249), STAT1 A488 (clone 4a; p-stat1, 560191), STAT3 A647 (clone49; p-stat3, 557815) and STAT5 PB (clone47: p-stat5, 560311) antibodies were utilized.

Techniques: Activation Assay, Expressing

Journal: Current Protocols

Article Title: Isolation and Molecular Profiling of Nuclei of Specific Neuronal Types from Human Cerebral Cortex and Striatum

doi: 10.1002/cpz1.70067

Figure Lengend Snippet: Antibodies and Dilutions Used in Basic Protocol 2

Article Snippet: TrueBlack ® Plus Lipofuscin Autofluorescence Quencher (Biotium, cat. no. 23014) PBS (see recipe) Wash buffer (chilled on ice, see recipe) DAPI buffer (chilled on ice, see recipe) AllPrep DNA/RNA FFPE Kit (Qiagen, cat. no. 80234) NeuN polyclonal chicken primary antibody (Millipore cat. no. ABN91) SATB2 monoclonal mouse primary antibody (Santa Cruz, cat. no. sc‐81376) BCL11B monoclonal rat antibody conjugated to FITC (Abcam, cat. no. ab123449) TLE4 monoclonal mouse antibody conjugated to PE (Santa Cruz, cat. no. sc‐365406) SLC1A3 monoclonal mouse primary antibody (Santa Cruz, cat. no. sc‐515839) IRF8 monoclonal mouse antibody conjugated to PE (Thermo Fisher Scientific, cat. no. 12‐9852‐82) OLIG2 polyclonal goat primary antibody (R&D Systems, cat. no. AF2418) Donkey anti‐Chicken A647 secondary antibody (Jackson ImmunoResearch, cat. no. 703‐605‐155) Donkey anti‐Mouse A594 secondary antibody (Invitrogen, cat. no. A‐21203) Donkey anti‐Mouse A488 secondary antibody (Invitrogen, cat. no. A‐21202) Donkey anti‐Goat A594 secondary antibody (Invitrogen, cat. no. A‐11058) Bucket with ice Refrigerating centrifuge with a swing‐out rotor for 1.5 ml tubes (e.g., Eppendorf, cat. no. 5810‐R) cooled to 4°C Shaker Nuclease‐free 1.5‐ml microcentrifuge tubes (e.g., USA Scientific, cat. no. 1615‐5500) Cell strainer snap cap (Corning, cat. no. 352235) FACS with at least a 4‐way sorting option and the ability to detect Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 647 and DAPI.

Techniques: Staining