Journal: PLoS ONE

Article Title: Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

doi: 10.1371/journal.pone.0073890

Figure Lengend Snippet: Immunofluorescence staining of microtubules (left) and PKA-RIIβ (right) in (A) retinoic acid-induced P19 neurons fixed at 1 day post-dissociation, (B) dissociated primary hippocampal neurons fixed at 1DIV, (C) dissociated primary dorsal root ganglion neurons fixed at 2DIV. All scale bars represent 20 µm.

Article Snippet: P19 cells (acquired from American Type Culture Collection, Manassas, VA) were maintained in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Biological Industries) and 1 mM sodium pyruvate.

Techniques: Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

doi: 10.1371/journal.pone.0073890

Figure Lengend Snippet: (A) Images of non-neuronal P19 cells transfected with plasmid overexpressing MAP2c-EGFP (left) or MAP2c-ΔRII-EGFP (right) for 20 hours, incubated with 1 µM of latrunculin-A for 4 hours, and fixed with formaldehyde. All images show signal from EGFP and all scale bars represent 20 µm. Quantification of average protrusion length (B) and average protrusion number per cell (C). * p < 0.05, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. More than 300 cells were analyzed for each constructs.

Article Snippet: P19 cells (acquired from American Type Culture Collection, Manassas, VA) were maintained in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Biological Industries) and 1 mM sodium pyruvate.

Techniques: Transfection, Plasmid Preparation, Incubation, Two Tailed Test, Construct

Journal: PLoS ONE

Article Title: Microtubule-Associated Type II Protein Kinase A Is Important for Neurite Elongation

doi: 10.1371/journal.pone.0073890

Figure Lengend Snippet: (A) Image of P19 cells transfected with plasmid overexpressing PKABD-Tub1 (left) or EGFP-Tub1 (right) constructs for 20 hours, incubated with 1 µM of latrunculin-A and 0.5 µM of taxol for 4 hours, and formaldehyde fixed. All scale bars represent 20 µm. Quantification of average protrusion length (B) or average protrusion number per cell (C). ** p < 0.01, two-tailed Student’s t-test. Error bars represent SEM from three independent repeats. More than 120 cells were analyzed for each constructs.

Article Snippet: P19 cells (acquired from American Type Culture Collection, Manassas, VA) were maintained in minimum essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Biological Industries) and 1 mM sodium pyruvate.

Techniques: Transfection, Plasmid Preparation, Construct, Incubation, Two Tailed Test

Journal: PLoS ONE

Article Title: Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

doi: 10.1371/journal.pone.0131674

Figure Lengend Snippet: A: IHC with anti-CD34 (red) and anti-BrdU (green) staining of hair follicles following 4-day BrdU pulse-chase in the Foxp1 fl/fl (WT) and K14-Cre; Foxp1 fl/fl ( cKO ) mice (P20-P23). The upper panel showed the timing of BrdU injection and sectioning. Abbreviations: Bu, bulge; HG, hair germ; DP, dermal papillae. Scale bars: 25 μm. B: Quantification of the number of BrdU + cells in the bulges of (A). The cKO hair follicles at P24 displayed extensive BrdU + cells in the hair germ and bulge cells, whereas the WT controls had few BrdU + cells in the identical regions (n = 3,4). *, p<0.05. C: IHC for hair follicles at P55 following 28-day BrdU pulse-chase. The upper panel showed the timing of BrdU injection and sectioning. Scale bars: 75 μm. D: Quantification of the percentages of LRC in the bulges of (C). Few label-retaining cells (LRC) were detected in the bulges of Foxp1 cKO mice. n = 4; *, p<0.05. E : NAC treatment and BrdU injection once a day from P23 to P26 enhanced cell proliferation of HFSCs in WT early anagen. Scale bar, 50μm. F: Quantification of the frequency of BrdU + cells in HFSCs in (E). *, p<0.05. G-H: Western blotting demonstrated a decrease of Foxp1 (G) and p19 ARF (H) protein levels within cKO hair follicles at anagen (P23). I: Down-regulation of p19 ARF transcripts within cKO anagen (P23) hair follicles relative to the WT as determined by qRT-PCR. J: S15 phosphorylated-p53 protein level was relatively decreased within cKO anagen (P23) hair follicles.

Article Snippet: The primary antibodies used were: anti-Foxp1 (Millipore, ABE68, USA, 1:1000); anti-phospho serine/threonine/tyrosine (Abcam, ab15556, UK, 1:1000); anti-p53 (Cell Signaling, 2524, USA, 1:1000); anti-p-p53 (Cell Signaling, 9284, USA, 1:1000); anti-β-actin (Santa Cruz, sc-81178, USA, 1:1000); anti-p19 ARF (Santa Cruz, sc-32748, USA, 1:1000).

Techniques: Staining, Pulse Chase, Injection, Western Blot, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Foxp1 Regulates the Proliferation of Hair Follicle Stem Cells in Response to Oxidative Stress during Hair Cycling

doi: 10.1371/journal.pone.0131674

Figure Lengend Snippet: A: Anti-Trx1 IHC confirming extensive expression of Trx1 in HFs at anagen (P23), catagen (P40) and telogen (P55). B: Interaction of Foxp1 and Trx-1 endogenous proteins in anagen HFs as determined by Co-IP of protein lysates. C: Interaction of Foxp1 and Trx-1 following ectopic expression of Foxp1-His and Trx1 in transfected HeLa cells. Cell lysates were immunoprecipitated by anti-Trx1 antibody and detected by anti-His antibody. D: Colocalization of Trx1-RFP and Foxp1-EGFP protein within the nuclei (blue, DAPI) of HaCat cells. E: Flow cytometry of DCFDA-stained HEK293T cells following transient transfection of the indicated constructs (2 μg Foxp1 and/or 2 μg Trx1 expressing vector) indicated that Foxp1 releases inhibition of Trx-1-mediated ROS accrual. F: Model for the mechanism by which Foxp1 regulates redox homeostasis during hair cycling. Foxp1 is located within nuclei under conditions of low oxidative stress. Foxp1 suppresses the function of the Trx1 protein in decreasing ROS levels, and then imposes cell cycle arrest through p19/p53 axis. Foxp1 is exported into the cytoplasm when the ROS levels approach a high threshold.

Article Snippet: The primary antibodies used were: anti-Foxp1 (Millipore, ABE68, USA, 1:1000); anti-phospho serine/threonine/tyrosine (Abcam, ab15556, UK, 1:1000); anti-p53 (Cell Signaling, 2524, USA, 1:1000); anti-p-p53 (Cell Signaling, 9284, USA, 1:1000); anti-β-actin (Santa Cruz, sc-81178, USA, 1:1000); anti-p19 ARF (Santa Cruz, sc-32748, USA, 1:1000).

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Flow Cytometry, Staining, Construct, Plasmid Preparation, Inhibition