Journal: PLoS ONE

Article Title: Interferon-γ Produced by Microglia and the Neuropeptide PACAP Have Opposite Effects on the Viability of Neural Progenitor Cells

doi: 10.1371/journal.pone.0011091

Figure Lengend Snippet: NPCs were treated with 100 ng/ml IFNγ for 2 days. *p<0.05 and ***p<0.001 comparing IFNγ treated vs. controls. ( A ) 10 µM BrdU was added to the cultures to label DNA and immunostaining done using anti-BrdU antibodies. Values are means±SD, n = 4. ( B ) Immunostaining using antibody against Ki67. Values are means±SD, n = 4. ( C ) Immunoblots. Cells were treated for 24 h. Note upregulation of p21 with no change in cyclinD1. Immunoprecipitation (IP) of p53 was performed prior to blotting. ( D ) RT-PCR for p21 was done as described. Typical experiment is shown and was repeated 3 times. ( E ) NPCs were treated with siRNA against p21 for 2 days followed by IFNγ for 24 h Upper panel, Immunoblots showing p-vimentin as a measure of cell proliferation. IFNγ decreased p-vimentin in control but not in p21-siRNA treated cells. Lower panel, BrdU labeling was decreased in control but not in p21-siRNA treated cells. ( F ) NPCs were treated with siRNA against p53 followed by IFNγ as above. IFNγ reduces cell proliferation in p53-siRNA treated cells. ( G ) p53 gene deleted mouse embryonic fibroblasts were treated with 10–100 ng/ml IFNγ for 2 days and cell viability determined as described in .

Article Snippet: Antibodies used were: cyclin D1 (1∶750) and Bax (1∶250) from Santa-Cruz (CA, USA), STAT1 (1∶400), p-STAT1 (1∶500); caspase-3 (1∶1000), cleaved caspase-3 (1∶1000); PARP (1∶2000), p53 (1∶1000), Bad (1∶1000), p-Bad (1∶500; phospho-Ser112), and Puma (1∶1000) all from Cell Signaling, Bcl2 (1∶400) and Bcl-xL (1; 1000) from BD Bioscience, IFNγR-2 (1∶3000; Abcam), p21 (1∶200; Millipore, MA, USA), p-vimentin (1∶2000; phospho-Ser55 Assay Designs, Ann Arbor, MI, USA), and β-actin (1∶2000, Sigma).

Techniques: Immunostaining, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Control, Labeling

Journal: PLoS ONE

Article Title: Interferon-γ Produced by Microglia and the Neuropeptide PACAP Have Opposite Effects on the Viability of Neural Progenitor Cells

doi: 10.1371/journal.pone.0011091

Figure Lengend Snippet: NPCs were treated with 100 ng/ml IFNγ for different times and analyzed as described in . *p<0.05 and **p<0.001 comparing IFNγ treated vs. controls. ( A ) Immunoblot shows increased levels of phospho-STAT1 (p-Stat1) after 0.5 h. ( B ) Immunostaining showed the presence of p-Stat1 in the nucleus after 2 h treatment. Scale bar, 10 µm. ( C ) Cell viability was determined by the MTT assay as described in . Values are means ±SD, n = 4. ( D ) Dose response curve. Cells were treated for 48 h and cell viability determined. Values are means ±SD, n = 4. ( E ) Cells were treated for 48 h and the number of nestin positive NPCs was analyzed. Values are means ±SD, n = 4. ( F ) Neurospheres were dissociated and about 5000 cells per well were incubated for 3days in absence or presence of 100 ng/ml IFNγ. The number of secondary neurospheres formed was counted. Values are means ±SD, n = 4.

Article Snippet: Antibodies used were: cyclin D1 (1∶750) and Bax (1∶250) from Santa-Cruz (CA, USA), STAT1 (1∶400), p-STAT1 (1∶500); caspase-3 (1∶1000), cleaved caspase-3 (1∶1000); PARP (1∶2000), p53 (1∶1000), Bad (1∶1000), p-Bad (1∶500; phospho-Ser112), and Puma (1∶1000) all from Cell Signaling, Bcl2 (1∶400) and Bcl-xL (1; 1000) from BD Bioscience, IFNγR-2 (1∶3000; Abcam), p21 (1∶200; Millipore, MA, USA), p-vimentin (1∶2000; phospho-Ser55 Assay Designs, Ann Arbor, MI, USA), and β-actin (1∶2000, Sigma).

Techniques: Western Blot, Immunostaining, MTT Assay, Incubation

Journal: PLoS ONE

Article Title: Interferon-γ Produced by Microglia and the Neuropeptide PACAP Have Opposite Effects on the Viability of Neural Progenitor Cells

doi: 10.1371/journal.pone.0011091

Figure Lengend Snippet: NPCs were treated for 24 h with 100 ng/ml IFNγ alone or in conjunction with 100 ng/ml PACAP. ( A ) Immunoblot. Cells were treated for 2 h. PACAP did not influence levels of p-Stat1 induced by IFNγ. ( B ) RT-PCR. SOCS-1 and –3 levels are not influenced by PACAP. IFNγ upregulated SOCS1. ( C ) Immunoblots. IFNγ increased whilst PACAP decreased levels of PUMA. PACAP also decreased active caspase-3 (17 kDa band). Typical blot is shown and was repeated three times. ( D ) Immunoblots. PACAP increased levels of p-Bad. Typical blot is shown and was repeated three times. ( E ) Effects of the kinase inhibitors on cell viability measured by MTT. 1 µM Gö6976 reduced the beneficial effect of PACAP whereas 10 µM H89 had no effect. Values are means ±SD, n = 4. *** p<0.005 for IFNγ vs. controls, and *p<0.0 for IFNγ+PACAP vs. IFNγ, and for GÖ+ IFNγ+PACAP vs. IFNγ+PACAP. ( F ) BrdU labeling. PACAP increases cell proliferation significantly in control but not in IFNγ treated cells. Values are means±SD, n = 4. * p<0.05 for PACAP vs control cells, n = 3.

Article Snippet: Antibodies used were: cyclin D1 (1∶750) and Bax (1∶250) from Santa-Cruz (CA, USA), STAT1 (1∶400), p-STAT1 (1∶500); caspase-3 (1∶1000), cleaved caspase-3 (1∶1000); PARP (1∶2000), p53 (1∶1000), Bad (1∶1000), p-Bad (1∶500; phospho-Ser112), and Puma (1∶1000) all from Cell Signaling, Bcl2 (1∶400) and Bcl-xL (1; 1000) from BD Bioscience, IFNγR-2 (1∶3000; Abcam), p21 (1∶200; Millipore, MA, USA), p-vimentin (1∶2000; phospho-Ser55 Assay Designs, Ann Arbor, MI, USA), and β-actin (1∶2000, Sigma).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Labeling, Control