A Sustainable Template for Mesoporous Zeolite Synthesis

A generalized synthesis of high-quality, mesoporous zeolite (e.g., MFI-type) nanocrystals is presented, based on a biomass-derived, monolithic N-doped carbonaceous template. As an example, ZSM-5 single crystals with desirable large-diameter (12–16 nm) intracrystalline mesopores are synthesized. The platform provides scope to optimize template dimensions and chemistry for the synthesis of a range of micro-/mesoporous crystalline zeolites in a cost-effective and highly flexible manner

Expression of Gli2 induces neurogenesis in P19 EC cells.

<p>(A–C): P19[Gli2] and P19[Control] cells were stained with Tuj1 and NF68 antibodies on day 6 or GFAP antibodies on day 10 of –RA differentiation. Nuclei were stained with Hoechst, scale bar is 30 µM. (<b>D</b>): Tuj1-, NF68- and GFAP-positive cells from (A)–(C) were counted in 10 random fields and expressed as a percentage of the total number of nuclei (10,000 cells; n = 4) (**p<0.01, n.s.  =  not significant). (<b>E</b>): NF68 immunoblot using total protein from day 6 differentiated P19, P19[Control] and two clonal populations of P19[Gli2] cell lines. P19 cells were differentiated in the presence of RA and served as a positive control. α-tubulin served as a loading control. (<b>F</b>): Total protein from –RA differentiating P19[Control] and P19[Gli2] cells was harvested on the days indicated, separated and immunoblotted with Gli2-specific antibodies. β-actin served as a loading control. Asterisk denotes non-specific binding of Gli2 antibodies.</p

Expression of Gli/EnR reduces expression of neuronal bHLH factors.

<p>Expression of indicated genes was assayed by QPCR analysis (Gli1, Gli3, n = 8; Gli2, MEF2C, n = 6; Gli/EnR, Sox2, Nestin, Ascl1, Neurog1, GFAP, n = 4; NeuroD, n = 3) by QPCR analysis. RNA from differentiating P19 (grey bars) and P19[Gli/EnR] cells (black bars) was harvested on days 0–6 +RA differentiation. Error bars represent +/− SEM from at least three biological replicas using two clonal populations (*p<0.05, **p<0.01).</p

Expression of Gli2 induces expression of neuronal bHLH factors.

<p>Expression of indicated genes was assayed by QPCR analysis, n = 4. RNA from differentiating P19[Control] (grey bars) and P19[Gli2] cells (black bars) was harvested on days 0, 2–6 and 9 of differentiation without RA. Error bars represent +/− SEM from at least two biological replicas using two clonal populations (*p<0.05, **p<0.01).</p

Gli2 activates the Ascl1 promoter.

<p>HEK-293 cells were transiently cotransfected with or without Gli2 and a construct containing the Ascl1 8 kb promoter driving the luciferase gene (Ascl1-luc) in ratios 2∶1, 4∶1 and 6∶1 relative to Ascl1-luc. Equal parts of Gli/EnR were transfected together with Gli2 at a ratio of 4∶1 relative to Ascl1-luc. Fold changes are relative to Ascl1-luc activity with the Ascl1-luc plasmid alone. Error bars represent +/− SEM from three biological replicas (*p<0.05, **p<0.01). No significant (n.s.) increase was observed in the presence of Gli/EnR.</p

Induction of neurogenesis in P19 EC cells by RA.

<p>P19 cells were differentiated using embryoid bodies in the presence of RA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019174#pone.0019174-JonesVilleneuve1" target="_blank">[35]</a>. (<b>A</b>): Formation of Tuj1-, and NF68- positive cells with neuronal morphology on day 6 and GFAP-positive cells with astrocyte morphology on day 10 of RA-induced (+RA) differentiation. Nuclei were stained with Hoechst, scale bar is 30 µM. (<b>B</b>): P19 EC cells fail to form Tuj1-, NF68- and GFAP- positive cells in the absence of RA (-RA) on the days indicated. Nuclei were stained with Hoechst, scale bar is 30 µM. (<b>C</b>): Tuj1-, NF68- and GFAP-positive cells from (A–B) were counted in 10 random fields and expressed as % of the total number of nuclei (3,000 nuclei). (<b>D</b>): The temporal pattern of expression of indicated genes during P19 EC differentiation +/−RA. Representative QPCR analysis is shown in which fold changes are relative to day 0. Error bars represent +/− SEM.</p

Summary of gene expression for P19 cells treated + and − RA, P19[Gli2] cells treated - RA, and P19[Gli/EnR] cells treated + RA.

<p>“++” means high upregulation as a result of overexpression, “+” means upregulation, “+/−” means no change, and “−” means downregulation of gene expression as compared to day 0. “N/A” means not applicable. For P19[Gli2] and P19[Gli/EnR] cell lines gene expression was compared to their respective control cell lines.</p><p>*Expression of Nanog was downregulated only in undifferentiated P19[Gli/EnR] cells;</p>#<p>Voronova and Skerjanc, unpublished observations.</p

Expression of Gli/EnR delays neurogenesis and decreases gliogenesis in P19 EC cells.

<p>Cells were differentiated using a monolayer procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019174#pone.0019174-Slack1" target="_blank">[46]</a> in the presence of RA. (<b>A–D</b>): Day 3 or day 6 differentiated P19[Gli/EnR] and P19 cells were stained with Tuj1- or NF68-specific antibodies. (<b>E</b>): Day 7 differentiated P19[Gli/EnR] and P19 cells were stained with GFAP-specific antibodies. Nuclei were stained with Hoechst, scale bar is 30 µM. (<b>F–H</b>): Tuj1-, NF68- and GFAP-positive cells from (A-E) were counted in 10 random fields and normalized with the number of nuclei (10,000 cells; n = 4), *p<0.05, **p<0.01, n.s.  =  not significant.</p

Oligonucleotide sequences of primers utilized for ChIP experiments.

<p>Oligonucleotide sequences of primers utilized for ChIP experiments.</p

Gli2 binds Ascl1 gene regulatory elements in P19 EC cells.

<p>(A–B): TRANSFAC (#M01037) Gli binding motif in forward and reverse direction, respectively. (<b>C</b>): Custom tracks of Ascl1 and Gli1 genes using UCSC genome browser (<a href="http://genome.ucsc.edu" target="_blank">http://genome.ucsc.edu</a>). Triangles designate the direction of transcription, and black boxes designate exons. The Ascl1 gene (+/− 100 kb) from mouse and human genomes was searched for conserved theoretical Gli binding as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019174#pone.0019174-Ovcharenko1" target="_blank">[53]</a>, which are designated as A–D. Their positions relative to the transcriptional start site (+1) are indicated as numbers. The known Gli binding site in the Gli1 gene is designated as A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019174#pone.0019174-Ikram1" target="_blank">[68]</a> (<b>D</b>): Comparison of mouse and human sequences of Ascl1 A–D sites from (C). The sequence of the Gli binding site (GBS) is marked in bold. (<b>E</b>): ChIP analysis showing enrichment by Gli2 antibodies of Ascl1 chromatin fragments corresponding to sites A–C, from (C). Sheared chromatin from day 4 -RA differentiated P19[Gli2] cells was immunopurified using Gli2-specific (black bars) or IgG non-specific (grey bars) antibodies. The Gli1 promoter served as a positive control. Percent chromatin input was calculated using QPCR analysis and primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019174#pone-0019174-t002" target="_blank">Table 2</a>. Error bars represent +/− SEM from three biological replicas (*p<0.05, **p<0.01, n.s.  =  not significant).</p