Reproducibility of olfactory fMRI activations in the OB and PC after MK801 injection.

<p>The data from all NHPs after MK801 injection were averaged to map the activations (p < 0.05, cluster size = 4) to define the ROIs of OB and PC. Within the OB and PC ROIs, color-encoded percentage fMRI signal changes were calculated from the averaged data over odd measurements (<b>A</b>) and from the averaged data over even measurements (<b>B</b>). The percentage change of fMRI signals for each voxel within the OB and PC for the two subsets are plotted against each other in (<b>C</b>) and (<b>D</b>). As shown in these figures, data points predominantly lie along their diagonal lines (slope = 1), indicating the fMRI signal changes from odd subsets are quantitatively similar to signal changes from even subsets. For the activations in OB (C), the cross correlation value is 0.91 (<i>p</i> = 1.41e^-42). For the activations in PC (D), the cross correlation value is 0.70 (<i>p</i> = 9.65e^-7). OB: olfactory bulb. PC: piriform cortex.</p

The impact of respiration rate on the olfactory responses in naïve NHPs.

<p>Data are from seven NHPs that did not receive any pharmacological agents. The 7 NHPs were separated into two groups based on their respiration rates. (<b>A & C</b>) Olfactory responses in the OB and PC from 4 NHPs with lower respiration rates of 19–25 breaths/min. (<b>B & D</b>) Olfactory responses in the OB and PC from 3 NHPs with higher respiration rates of 29–35 breaths/min. The difference in respiration rate from these 2 groups of NHPs has no effect on the olfactory responses in the OB and PC.</p

Definition of the 2 ROIs and the olfactory fMRI responses in these ROIs before and after MK801 injection.

<p>(<b>A</b>) The color-encoded ROIs were superimposed on the 3 consecutive axial gradient-echo EPI images. Purple: olfactory bulb (OB); red: piriform cortex (PC). (<b>B & C</b>) Olfactory response in the OB and PC in the different 1-h periods of 4-h experiment session (mean ± SD, n = 4). Red bars: 1-min odor stimulation. In the OB (<b>B</b>), robust olfactory fMRI response was observed before MK801 injection, and MK801 increased the response. In the PC (<b>C</b>), no robust olfactory response was observed before MK801 injection. After MK801 injection, robust and repeatable olfactory responses were observed. (<b>D & E</b>) Strengths of the olfactory responses during the 4-h experimental session (mean ± SD, n = 4). MK801 increased the response amplitudes in the OB and PC. The asterisks ‘*’ indicate that the strengths of response were significantly larger than the strengths during the first hour before MK801 delivery (paired <i>t</i>-test, all p<0.04).</p

Olfactory fMRI activations before and after MK801 injection.

<p>The data from all NHPs were averaged to map the activations (p < 0.05, cluster size = 4). (<b>A</b>) Olfactory fMRI Activations <i>before</i> MK801 injection. Color-encoded activations (red/yellow) were observed in the consecutive 3 slices where olfactory bulb is located. <b>(B)</b> Olfactory fMRI activations <i>after</i> MK801 injection. Color-encoded activations were observed in the consecutive 3 slices where olfactory bulb and piriform cortex are located. Green arrows: activations in the piriform cortex. OB: olfactory bulb.</p

SUMOylation of Sox2 represses Nanog expression.

<p>(A) Covalent modification of Sox2 by Sumo1 at Lysine 247. Wild-type Sox2 and mutant Sox2 K247R were coexpressed with HA-Sumo1 and HA-Ubc9. (B) qPCR analysis of Nanog mRNA in response to various levels of SUMOylated Sox2. (C) Western blot analysis of Nanog in F9 EC cells under a varying status of SUMOylated Sox2. (D) Covalent modification of Sox2 with Sumo1 inhibits the transcriptional activity of the Nanog proximal promoter. Transcriptional activities of the Nanog proximal promoter (−230 to +50 bp relative to the transcription start site) in response to various levels of SUMOylated Sox2 were determined by dual-luciferase reporter assays. qPCR data were normalized to <i>GAPDH.</i> Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.</p

SUMO E3 ligases PIAS proteins mediate substrates-specific SUMOylation and regulate Nanog transcription.

<p>(A and B) Pias2 and Pias3 promote SUMOylation of Oct4 and Sox2, respectively. NIH3T3 cells were transfected with various combinations of plasmids as indicated. SUMOylated Oct4 and Sox2 was enriched by CoIP using an anti-Sumo1 antibody, and detected by western blot with anti-Oct4 and anti-Sox2 antibodies, respectively (upper panel). (C and D) <i>Nanog</i> transcription is up-regulated by Pias3, but down-regulated by Pias2. Transfection of F9 EC cells with various combinations of plasmids as indicated. The levels of <i>Nanog</i> transcripts were normalized against <i>GAPDH</i> expression. Data are presented as the mean +/− SD and are derived from three independent experiments. *: p<0.05; **: p<0.01. CoIP: co-immunoprecipitation; WB: western blot.</p

SUMOylation of Oct4 enhances Nanog expression.

<p>(A) Oct4 is modified by Sumo1 at Lysine 118. Wild-type Oct4 or the SUMO receptor site mutant Oct4 K118R was expressed in combination with HA-Sumo1 and HA-Ubc9 in F9 EC cells. (B) qPCR analysis of <i>Nanog</i> mRNA in response to various levels of SUMOylated Oct4. The levels of the transcripts were normalized against control empty vector transfection. (C) Western blot analysis of Nanog in F9 EC cells under a varying SUMOylation status of Oct4. (D) SUMOylation of Oct4 enhances the <i>Nanog</i> proximal promoter transcription. Transcriptional activities of the <i>Nanog</i> promoter (−230 to +50 bp relative to the transcription start site) in response to various levels of SUMOylated Oct4 were determined by dual-luciferase reporter assays. qPCR data were normalized to <i>GAPDH.</i> Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.</p

SUMOylation impairs the protein-protein interaction between Oct4 and Sox2.

<p>NIH3T3 cells were cotransfected with various combinations of Oct4/Oct4 K118R, Sox2/Sox2 K247R, HA-Sumo1 and HA-Ubc9 expression plasmids as indicated. Cell extracts were respectively co-immunoprecipitated with anti-Oct4 and anti-Sox2 antibody-coated affinity beads. Whole-cell lysates (input) and immunoprecipitated proteins were separated by 12% SDS-PAGE, followed by western blot with anti-Sox2, anti-Oct4, or anti-GAPDH antibodies. Western blot images were analyzed using Image J. (A) The protein-protein interaction between wild-type Oct4 and Sox2, the relative band intensity values of samples to controls were presented in bar histogram. (B) The protein-protein interaction between wild-type Oct4/Sox2 and mutant Sox2 K247R/Oct4 K118R, the relative band intensity values of samples to controls were presented in bar histogram. CoIP: co-immunoprecipitation; WB: western blot.</p

SUMOylation represses Nanog expression in F9 embryonal carcinoma cells.

<p>(A) Endogenous Sumo1 expression in control and Sumo1-knockdown F9 EC cells. After 48 hours post-transfection with a Sumo1-specific shRNA construct (sh-Sumo1) or negative control (Vector and sh-scramble), Sumo1 expression was determined by qPCR and western blot. (B) Endogenous Ubc9 expression in control and Ubc9-knockdown F9 EC cells. After 48 hours post-transfection with an Ubc9-specific shRNA construct (sh-Ubc9) or negative control (Vector and sh-scramble), Ubc9 expression was determined by qPCR and western blot. (C) Overexpression of Sumo1 and/or Ubc9 in F9 EC cells. F9 EC cells were transfected with pCMV-HA-Sumo1, pCMV-HA-Ubc9 and empty vector as indicated, Sumo1 and Ubc9 mRNA levels were detected by qPCR respectively. (D) qPCR analysis of Nanog expression in F9 EC cells in response to knockdown of Sumo1/Ubc9. (E) qPCR analysis of Nanog expression in F9 EC cells in response to overexpression of <i>Sumo1/Ubc9.</i> (F) Endogenous <i>Nanog</i> protein in F9 EC cells were determined by western blot after transient transfection with the indicated constructs. (G) Transcriptional activity of the <i>Nanog</i> proximal promoter in response to SUMOylation. After 48 hours post-transfection with the indicated plasmids, luciferase activity was determined and normalized against control empty vector transfection. qPCR data were normalized to <i>GAPDH.</i> Data are presented as the mean +/− SD and are derived from three independent experiments. *, p<0.05; **, p<0.01; WB: western blot.</p

SUMOylation does not alter the subcellular localization of Oct4 and Sox2.

<p>(A) Subcellular localization of Oct4 and Oct4K118R. F9 EC cells were cotransfected with red fluorescent protein tagged Oct4/Oct4 K118R plasmids and HA-Sumo1 or HA-Ubc9. There is no obvious difference in the subcellular localization of Sumo1-modified and unmodified Oct4. (B) The distribution of Sox2 and Sox2 K247R in F9 EC cells. Cotransfection of F9 EC cells with pDsRed-<i>Sox2</i>/pDsRed-<i>Sox2</i> K247R and HA-Sumo1 or HA-Ubc9. Both SUMOylated Sox2 and unmodified Sox2 K247R localize in the nuclei. Nuclei were stained with DAPI (blue). Cells were observed and photographed under a Nikon confocal microscope at ×400 magnification.</p