Position of the identified PAX9 mutation.

<p>(A) Locations of P20L and previously reported missense variants are shown with arrows above and below the PAX9 diagram, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref012" target="_blank">12</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref025" target="_blank">25</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.ref031" target="_blank">31</a>]. Reported pathogenic variants with nonsense mutations or frameshifts are omitted. NSD, N-terminal subdomain of the paired domain; CSD, C-terminal subdomain of the paired domain. (B) Modeled pair-domain structure of the wild-type (blue and red with green backbone) and P20L (yellow) PAX9. P20L mutation results in Van del Waals clashes with the carbonyl oxygen of Leu²¹ (red disc) and the side chain of Pro⁶⁸ (brown disc). This results in a slight displacement of Pro⁶⁸. DNA is shown in pink with a gray surface.</p

Electrophoretic mobility shift assay of PAX9^WT and PAX9^P20L.

<p>Nuclear extract was prepared at 48 h after transfection with PAX9-Myc-expressing (WT or P20L) or 1st ATG-deleted plasmids (Null), and analyzed using the CD19-2(A-ins) probe. Ectopic expression of PAX9-Myc and integrity of nuclear extract were confirmed with immunoblotting (bottom). Excessive amount of the non-biotinylated DNA eliminated the mobility shift signals in PAX9^WT, whereas addition of anti-Myc antibody resulted in a super-shift, confirming the specificity of the bipartite interaction. No detectable degree of signal shift was observed in PAX9^P20L. No NE, no nuclear extract added; L probe, biotin-labelled probe; NL probe, non-labelled probe.</p

Pedigree with tooth genesis involving three patients.

<p>(A) Proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>), her father (ID 7) and brother (ID 10) are affected (indicated with filled symbols), while her mother is unaffected (indicated with an open symbol). The inheritance pattern is consistent with autosomal dominance. The three affected members invariably show C/T heterozygosity at the second position of the Pro²⁰ codon, while the unaffected member shows C/C homozygosity. (B) Radiogram of the proband (patient ID 9 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186260#pone.0186260.t001" target="_blank">Table 1</a>). Missing teeth other than 3rd molars are indicated with asterisks.</p

Luciferase reporter assay using the <i>BMP4</i> promoter region as a cis element.

<p>Activity of firefly luciferase expressed from the reporter vector was normalized with <i>Renilla</i> luciferase, and shown as arbitrary units (mean ± SEM). Experiments were repeated three times with independent transfections, and for each experiment luciferase activity was measured in triplicate. Protein samples were prepared from aliquots of transfected cells, and ectopic PAX9 expression was verified by immunoblotting. An alleviating effect of P20L on transactivation was supported statistically, while that of A240P was not, as shown above the graph.</p

Tuning Lewis Acidity of Metal–Organic Frameworks via Perfluorination of Bridging Ligands: Spectroscopic, Theoretical, and Catalytic Studies

The Lewis acidity of metal–organic frameworks (MOFs) has attracted much research interest in recent years. We report here the development of two quantitative methods for determining the Lewis acidity of MOFsbased on electron paramagnetic resonance (EPR) spectroscopy of MOF-bound superoxide (O₂^•–) and fluorescence spectroscopy of MOF-bound <i>N</i>-methylacridone (NMA)and a simple strategy that significantly enhances MOF Lewis acidity through ligand perfluorination. Two new perfluorinated MOFs, Zr₆-fBDC and Zr₆-fBPDC, where H₂fBDC is 2,3,5,6-tetrafluoro-1,4-benzenedicarboxylic acid and H₂fBPDC is 2,2′,3,3′,5,5′,6,6′-octafluoro-4,4′-biphenyldicarboxylic acid, were shown to be significantly more Lewis acidic than nonsubstituted UiO-66 and UiO-67 as well as the nitrated MOFs Zr₆-BDC-NO₂ and Zr₆-BPDC-(NO₂)₂. Zr₆-fBDC was shown to be a highly active single-site solid Lewis acid catalyst for Diels–Alder and arene C–H iodination reactions. Thus, this work establishes the important role of ligand perfluorination in enhancing MOF Lewis acidity and the potential of designing highly Lewis acidic MOFs for fine chemical synthesis

Tuning Lewis Acidity of Metal–Organic Frameworks via Perfluorination of Bridging Ligands: Spectroscopic, Theoretical, and Catalytic Studies

The Lewis acidity of metal–organic frameworks (MOFs) has attracted much research interest in recent years. We report here the development of two quantitative methods for determining the Lewis acidity of MOFsbased on electron paramagnetic resonance (EPR) spectroscopy of MOF-bound superoxide (O₂^•–) and fluorescence spectroscopy of MOF-bound <i>N</i>-methylacridone (NMA)and a simple strategy that significantly enhances MOF Lewis acidity through ligand perfluorination. Two new perfluorinated MOFs, Zr₆-fBDC and Zr₆-fBPDC, where H₂fBDC is 2,3,5,6-tetrafluoro-1,4-benzenedicarboxylic acid and H₂fBPDC is 2,2′,3,3′,5,5′,6,6′-octafluoro-4,4′-biphenyldicarboxylic acid, were shown to be significantly more Lewis acidic than nonsubstituted UiO-66 and UiO-67 as well as the nitrated MOFs Zr₆-BDC-NO₂ and Zr₆-BPDC-(NO₂)₂. Zr₆-fBDC was shown to be a highly active single-site solid Lewis acid catalyst for Diels–Alder and arene C–H iodination reactions. Thus, this work establishes the important role of ligand perfluorination in enhancing MOF Lewis acidity and the potential of designing highly Lewis acidic MOFs for fine chemical synthesis

GRPs were highly enriched for gene expression of stemness, IGF1, and IGF1R.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1, which are stemness genes in PC9 or HCC827 parental cells and GRPs. <b>B.</b> Quantitative RT-PCR was performed with primers specific for IGF1 and IGF1R in PC9 or HCC827 parental cells and GRPs. Data were normalized to actin expression. *p<0.01, **p<0.001, ***p<0.0001.</p

IGF1R was phosphorylated on hypoxic GRPs, and knockdown of IGF1 decreased the population of CD133- and Oct4-positive hypoxic GRPs.

<p><b>A.</b> Quantitative RT-PCR was performed with primers specific for IGF1 in PC9 or HCC827 parental cells, GRPs, and hypoxic GRPs. Data were normalized to actin expression. **p<0.001. <b>B.</b> PC9 cells, grown on Lab-Tek chamber slides with or without 1 µM gefitinib and under normoxic or hypoxic conditions for 72 h, fixed, and incubated with the primary antibodies against phosphorylated IGF1R (pIGF1R) and then with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained using an Axioplan 2 system imaging with AxioVision software. Images used to compare PC9 parental cells, GRPs, and hypoxic GRPs were acquired with the same instrument settings and exposure times, and were processed similarly. The number of pIGF1R-positive cells was counted, and the ratio of these cells was calculated in five fields in each experiment. **p<0.001. <b>C.</b> IGF1 expression was knocked down in PC9 or HCC827 hypoxic GRPs by using small interfering RNA (siRNA) in Lab-Tek chamber slides. Immunofluorescence staining for pIGF1R, CD133, or Oct4 was then performed. Two specific siRNAs and one non-specific control were used. The numbers of pIGF1R-, CD133- and Oct4-positive cells were counted, and the ratio of these cells was calculated in five fields for each experiment. **p<0.001, *p<0.01.</p

Sphere-forming ability of GRPs was upregulated and hypoxia increased this capacity.

<p>PC9 parental cells, GRPs, and hypoxic GRPs were prepared at densities of 2.5×10³ cells per 2 mL per well in serum-free media supplemented with growth factors and seeded into 6-well ultra-low attachment plates. PC9 parental cells and GRPs were incubated under normoxic conditions, while PC9 hypoxic GRPs were grown under hypoxic conditions. Culture medium was fed every 3 days. The number and size of spheres were recorded and immunofluorescence was performed 7 days after the start of the culture period. Spheres were fixed and incubated with primary antibodies against CD133, Oct4, or phosphorylated IGF1R (pIGF1R), and then with secondary antibody labeled with Alexa Fluor 594 goat anti-mouse IgG (red) or Alexa Fluor 488 goat anti-rabbit IgG (green). Cell nuclei were stained with DAPI (blue). Images were obtained on an Axioplan 2 imaging system with AxioVision software. <b>A.</b> The number of spheres was significantly increased in PC9 GRPs compared to in parental cells, and was further increased in PC9 hypoxic GRPs. *p<0.01, # p<0.05. <b>B.</b> Sphere size of PC9 hypoxic GRPs was significantly greater than that of parental cells. # p<0.05. <b>C.</b> Immunofluorescent images of control cells of PC9 (left) and spheres of GRPs (right) for CD133, Oct4, and pIGF1R.</p

Tumor incidence of parental PC9 cells and normoxic and hypoxic GRPs transplanted into NOG mice.

<p>GRPs; Gefitinib resistant persisters.</p><p>NOG mice; NOD/Shi-scid/IL-2Rcnull (NOG) mice.</p>#<p>Tumor incidence was increased significantly in hypoxic GRPs group as compared with parental cell group with 1×10¹ cells/injection, p<0.05.</p><p>To evaluate the <i>in vivo</i> tumorigenic potential, 1×10 cells or 1×10² cells of parental PC9 cells or normoxic and hypoxic PC9 GRPs were mixed with Matrigel and injected into both flanks of NOG mice. Tumor formation was evaluated 33 days after injection.</p