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Results: 9

1.
Figure 6.

Figure 6. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

SS18 and CREST switch during artificial neuron development. Human fibroblasts were infected with lentivirus containing miR-9/9* and miR-124. Eighteen days later, mRNA was isolated. qRT-PCR analysis shows the switch from SS18-containing BAF complexes in fibroblasts to predominantly CREST-containing complexes after miR-9/9* and miR-124 addition to human fibroblasts. After the switch, the transcript profile is similar to that of an adult human brain sample. Data are shown ± SEM of three separate neuronal inductions. *p < 0.05, Student's t test.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
2.
Figure 9.

Figure 9. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

Summary of ordered events in npBAF to nBAF complex switching. A, The npBAF to nBAF switch involves three subunits regulated by miR9/9* and miR-124. Combinatorial assembly of npBAF and nBAF subunits could create up to 36 distinct npBAF or nBAF complexes. B, In a negative cascade network motif, downstream genes are sequentially repressed and activated when their regulator reaches the relevant threshold. The conversion of ES cells to neurons illuminates the sequential repression and activation of the genes of the triple-negative miRNA/chromatin switch, which is a negative cascade network. BAF53b is under independent control for transcription, but the presence of BAF53a will drive BAF53b from the BAF complex and destabilize 53b protein (B.T.S, G.R.C, unpublished results).

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
3.
Figure 7.

Figure 7. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

SS18 knock-down causes a G2-M-phase block and reduced rate of S-phase entry in ES and NS cells and results in failure of NS cells to self-renew. A, ES cell cycle analysis. Bottom left, G1; top, S-phase (DNA synthesis); bottom right, G2-mitosis. Lentivirus infection with shScramble shSS18#1. shSS18 knock-down causes G2-M-phase block and slower progression into the S-phase. SS18 protein quantification of ES cells. Data are representative of three separate experiments done in duplicate. B, Reduction of SS18 protein with shSS18 slows proliferation rate of ES cells. C, Neurosphere NS cell-cycle analysis. Infection with shSS18#1 caused a G2-M block and reduced S-phase entry. Shown is SS18 protein quantification of NS cells. The SS18 protein level was reduced ∼25%, indicating SS18 haploinsufficiency. Data are shown ± SEM. D, Secondary neurosphere formation is blocked by SS18 knock-down, indicating failure of NS cell self-renewal. Neurosphere NS cell analysis is representative of two separate experiments done in duplicate. Scale bar, 100 μm.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
4.
Figure 5.

Figure 5. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

Kinetics of transcriptional induction and repression of nBAF and npBAF genes, miRNAs, cell-type marker genes, and neurogenic transcription factors after neural induction of ES cells. A, qRT-PCR analysis of mRNA and microRNAs during neural induction time course. Green panel denotes neural progenitor stage, red panel denotes neuron stage, and vertical line delineates time of cell cycle exit. B, Protein analysis by Western blot. BAF45c is the first nBAF subunit to be activated and BAF53b is the last to be activated. Data are representative of three separate ex vivo differentiations. C, BAF45c is activated independent of RA induction. BAF45b and BAF53b are not activated without RA induction. D, BAF250a, 250b, BAF60a, BAF60b, and BAF60c expression kinetics. qRT-PCR is presented as a percentage of the maximum transcript level ± SEM of three separate ex vivo differentiations.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
5.
Figure 4.

Figure 4. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

Kinetics of induction and loss of nBAF and npBAF subunits after neural induction of ES cells. A, Schematic of neural induction of ex vivo ES cell-to-neuron differentiation time course. Open arrowhead denotes cell aggregate dissociation and transfer of neural progenitors to neural differentiation media and poly-ornithine-, laminin-, and fibronectin-coated plates. B, SS18 protein intensity decreases when cells differentiate into Tuj+ neurons. CREST is first detected on day 9 in Tuj+ neurons with rapidly increasing intensity as the cells become more mature neurons. BAF53a/45a decrease in neurons and BAF53b/45b are activated only in Tuj+ neurons. BAF53a colocalizes with Nestin+ NS/progenitor cells. BAF45c is activated before BAF45b and before cells becoming Tuj+. C, Proliferation measured by EdU incorporation during the switch from Nestin+ NS/progenitors to Tuj+ neurons. Cell cycle exit corresponds with the timing of the npBAF to nBAF switch. Cells were pulsed with EdU for 2 h before being fixed. Data are shown ± SEM. Images and EdU staining are representative of three separate ex vivo differentiations.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
6.
Figure 3.

Figure 3. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

SS18 and CREST remain associated with Brg up to 5 m urea. A, Denaturation analysis of BAF complexes in ES cells. SS18 and BAF53a remain associated with Brg at 5 m urea, whereas BAF155 comes off in 2.5 m urea. Nuclear extracts were prepared from E14 ES cells as described in the text. Nuclear extracts were subjected to different concentrations of urea for 10 min at 25°C before IP, diluted 10× with IP buffer, incubated with anti-Brg (G7) antibodies overnight at 4C. Brg co-IP proteins were isolated, electrophoresed, and probed with antibodies to the labeled proteins. B, Denaturation analysis of npBAF complexes in E10.5 neural progenitors or E18.5 neurons. npBAF containing SS18 in E10.5 nuclear extracts switches to nBAF containing CREST in E18.5 neurons. SS18 remains associated with Brg to 5 m urea in neural progenitors, whereas CREST remains associated with Brg to 5 m urea in neurons. No CREST is detected at E10.5 and very little SS18 is detected at E18.5. TBP is blotted as a loading control. C, Denaturation analysis of nBAF complexes in P1.5 brain extracts. CREST remains associated with Brg up to 5 m urea, whereas BAF170 comes off at 2.5 m urea. Western blots are representative of three separate protein purifications from the respective cell types.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
7.
Figure 1.

Figure 1. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

Identification of novel BAF subunits SS18 and CREST. A, Table of mass-spectrometric analysis of proteins co-purified with Brg/Brm from mouse ES cells, MEFs, and newborn P0 whole brain. Shown is the percent coverage based on unique peptides. B, Glycerol gradient (10–30%) analysis of E10.5 and E18.5 mouse brain cortical nuclear extracts. SS18 but not CREST cosediments with npBAF complexes at 2MDa in E10.5 mouse brain nuclear extracts, whereas CREST cosediments with nBAF complexes in E18.5 mouse brain nuclear extracts. The Polycomb PRC2 complex protein Ezh2 was blotted as a control and peaks in ∼1 MDa fractions. *From other studies, these proteins are not found in pure undifferentiated ES cells nor MEFs. The presence of these proteins in ES cells could be from spontaneously differentiating cells in the ES cultures and neural cells in the MEFs. Glycerol gradients are representative of two separate protein purifications from each developmental time point.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
8.
Figure 2.

Figure 2. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

SS18 and CREST are mutually exclusive, nonexchangeable subunits of npBAF and nBAF complexes, respectively. A, Co-IP from E15.5 brain cortical extracts; Brg co-IPs SS18 and CREST whereas CREST does not co-IP SS18 and SS18 does not co-IP CREST. Black arrowheads mark the SS18 and CREST proteins. Empty arrowhead marks SS18 Ab cross-reacting with CREST (CREST runs above SS18 on these Western blots). *Immunoglobin heavy chain. B, Immunostaining on cross sections of E12.5 spinal cords. SS18 is expressed specifically in neural progenitors in ventricular zone (VZ) and is replaced by the homologous subunit CREST in postmitotic neurons in the postmitotic zone (PMZ). Scale bar, 250 μm. C, Mutual exclusive expression pattern of SS18 and CREST as determined by costaining primary mouse E15.5 cortical cells cultured for 4 d in vitro with neural markers. SS18 is expressed in Nestin+ NS/progenitors, whereas CREST is specifically expressed in Tuj+ neurons. Scale bar, 20 μm. D, Quantification of staining in C. Data are ± SD of three fields of view each, with ∼100 cells each field.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.
9.
Figure 8.

Figure 8. From: Kinetic Analysis of npBAF to nBAF Switching Reveals Exchange of SS18 with CREST and Integration with Neural Developmental Pathways.

SS18 expression prevents KCl-induced dendritic outgrowth. A, Total dendrite length. Primary E18.5 cortical neurons were isolated, transfected with iresGFP, SS18-iresGFP, or CREST-iresGFP where indicated, cultured for 6 d with 30 mm KCl for the last day where indicated. Control vector and CREST overexpression do not affect dendrite outgrowth in response to KCl depolarization. SS18 expression reduces activity-dependent dendritic outgrowth in response to KCl depolarization. B, Number of branch points per cell, a measure of dendritic arbor complexity, is also reduced. The average values are from three independent experiments, with 50–75 GFP+ neurons scored per condition per experiment. Data are shown ± SEM. *p < 0.02, **p < 0.002, ***p < 0.0005, Student's t test. C, Representative images of neurons with or without KCl. D, Nuclear extracts of ES cells infected with V5-tagged SS18 and CREST integrate into BAF complexes, as evidenced by co-IP with Brg and BAF155. E, Exogenous SS18 and CREST protein in cortical neurons is nuclear and SS18 expression in neurons decreases CREST protein levels after depolarization with KCl. Arrow points to neuron nuclei with decreased CREST. Images are of KCl-depolarized samples. F, Quantification CREST protein level expression (average fluorescence intensity in nuclei). Forced SS18 expression causes significant decrease in CREST protein with depolarization. The average values are from two independent experiments, with 20–30 GFP+ neurons scored per condition per experiment. Dara are shown ± SEM. *p < 0.02, **p < 0.002, ***p < 0.0005, Student's t test. Scale bar in C, 50 μm; in E, 20 μm.

Brett T. Staahl, et al. J Neurosci. 2013 June 19;33(25):10348-10361.

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