Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 1. Deletion of Sox2 leads to proliferation of neonatal IPCs. A–F, Triple staining of myosin VI, Sox2, and EdU in Fgfr3iCreER; Sox2/ control (A–C) and Fgfr3iCreER; Sox2loxP/loxP experimental (D–F) samples given tamoxifen at P0 and P1 at theHClayer(A,D)andSClayer(B,E).CandFareartificialcross-sectionimagesintheYZplane.ArrowsinEandFpointtothesame EdU/Sox2-negative IPC. Dashed lines in C and F represent the basilar membrane. G–H, Control (G) and experimental (H–H) sampleswerestainedwithmyosinVI,EdU,andBrdU.GandHareimagestakenattheHClayer,andHattheSClayer.ArrowinH showsanEdU/BrdUIPC.I,QuantificationofEdUIPCsintheentirecochleaofexperimentalsamplesgivenTMXatP0andP1, EdU at P2, and analyzed 6 h later or at P4.**p 0.01 (n 3). Scale bars, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Staining, Control, Membrane

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 2. Deletion of Sox2 results in loss of p27Kip1 expression in neonatal IPCs without cell fate change. A, Quantification of Sox2- negativeandp27Kip1-negativeIPCsatP2intheentirecochleaofFgfr3iCreER;Sox2loxP/loxPexperimentalmiceinjectedwithtamoxifenatP0 andP1.B–B,TriplestainingofSox2,p27Kip1,andEdUincochlearsamplesfromexperimentalmice.ArrowspointtothesameEdU/ Sox2-negative/p27Kip1-negativeIPC.ArrowheadspointtothesameSox2-negativeIPCthatmaintainedfaintexpressionofp27Kip1 and was EdU-negative, which is also visualized at a higher magnification (inset in B). C–D, Double labeling of Sox2 and Prox1 in control Fgfr3iCreER;Sox2/(C–C)andexperimentalFgfr3iCreER;Sox2loxP/loxP(D–D)samples.ImageswerevisualizedatconfocalYZplane. MoreIPCswerepresentinexperimental(dashedcircleinD)thanincontrolgroups.Scalebars,20m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Expressing, Labeling, Control

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 3. Sox2 ablation causes a loss of p27Kip1 expression and S phase reentry of juvenile IPCs. A–B, Fgfr3iCreER; Sox2/

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Expressing

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure4. Sox2-negativeIPCsinjuvenilemicecannotcompletetheentirecellcycleandmaintainedaSCfate.A–B ,TriplestainingofSox2,pH3andEdUinsamplesfromFgfr3iCreER;Sox2loxP/loxP

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques:

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure7. Proliferatingp27 Kip1-nullPCsmaintainProx1andSox2expression.A–A,Double labeling of Sox2 and p27 Kip1 at P2 in Prox1CreER/; p27loxP/loxP (experimental) samples given tamoxifenatP0andP1.ArrowspointtothesameSox2/p27 Kip1-negativeIPC.B–B,Cross- section staining of EdU and Prox1 at P2. Arrows point to the same Prox1/ EdU PC. Similar results were observed at P4. Scale bars, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Labeling, Staining

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure 8. Long-term effects caused by proliferation of neonatal IPCs. A–B, Whole-mount image of calbindin HCs in Fgfr3iCreER; Sox2/ control (A, A) and Fgfr3iCreER; Sox2loxP/loxP experimental (B, B) groups. Arrow in B indicates 3 missed IHCs. C, Projection image of the rectangular area in B showing loss of OHCs. D, D, Image of TUNEL and calbindin labeling in experimental mice at P16. Arrows indicate dying cells. Scale bars: B, 200 m; C–D, 20 m.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Control, TUNEL Assay, Labeling

Journal: Journal of Neuroscience

Article Title: Regulation of p27Kip1 by Sox2 Maintains Quiescence of Inner Pillar Cells in the Murine Auditory Sensory Epithelium

doi: 10.1523/jneurosci.0686-12.2012

Figure Lengend Snippet: Figure9. Sox2regulatesp27 Kip1invitro.A,Schematicoftheluciferaseconstructusedinthereporterassay.Theblueregionis a 3.8 kb putative p27kip1 promoter fragment. B–D, The effect of Sox2 overexpression on p27Kip1 transcriptional activity was measured in MEF (B), HeLa (C), and HEK cells (D). All values were normalized to the negative control empty vector (EV), then compared with the positive control E2F1, or Sox2 overexpression. Minimal luciferase activity was detected when a promoter-less luciferase vector was used (empty-luc), with no increases occurring in the presence of Sox2 or E2F1. E, Schematic of the p27- luciferase plasmid and amplicon location. F, qPCR data from ChIP experiments performed in MEF cells transfected with p27- luciferaseplasmidandSox2.Asignificantenrichmentofamplicon7(1400bpupstreamofLuciferaseORF)wasobservedwhen the Sox2 antibody was used for ChIP. *p 0.05.

Article Snippet: Next DNA/protein complexes were immunoprecipitated using 2 l of ChIP-formulated Sox2 antibody (Cell Signaling Technology, 5024).

Techniques: Over Expression, Activity Assay, Negative Control, Plasmid Preparation, Positive Control, Luciferase, Amplification, Transfection