IL-4-induced STAT6 signaling in parental and mutant cells.

<p>Si, AnxA2<i>kd</i>, or AnxA5<i>kd</i> cells were transiently transfected with p4xSTAT6-Luc2P and treated with 0, 1, or 10 ng/mL IL-4 for 24 hours, after which Luciferase activity was measured. Each bar represents mean Luciferase activity ± SEM normalized to vehicle control, n = 3. *, ***represent statistically significant difference from Si control, *p<0.05, **p<0.01; + represents statistically significant difference compared to Siren at same IL-4 concentration, p<0.001.</p

Effects of osteogenic differentiation on expression of AnxA2 and AnxA5.

<p>qPCR analysis of (A) <i>AnxA2</i> or (B) <i>AnxA5</i> expression in undifferentiated Si cells at day 0 and in cells cultured in maintenance or osteogenic media 7, 14 and 21 days. Each bar represents mean transcript normalized to α-tubulin ± SEM, n = 3–5. *, **represent statistically significant difference from maintenance control at same time point, *p<0.05, **p<0.01; + represents statistically different from same media composition on day 0, p<0.05.</p

Effects of <i>AnxA2</i> and <i>AnxA5</i> knockdown on expression of genes associated with osteogenic differentiation.

<p>qPCR analysis of (A) <i>Runx2</i>, (B) <i>Sp7</i>, (C) <i>Col1a1</i>, (D) <i>Ibsp,</i> and (E) <i>Bglap</i> expression in undifferentiated Si, <i>AnxA2</i>kd and <i>Anx5</i>kd cells (day 0) and in cells cultured in differentiation medium for 7, 14 and 21 days. Each bar represents mean transcript normalized to α-tubulin ± SEM, n = 3. *, **, ***represent statistically significant difference from Si at same time point, *p<0.05, **p<0.01, ***p<0.001; + represents statistically significant difference from same genotype on day 0, p<0.05.</p

Decreased proliferation in <i>AnxA2</i>kd and <i>AnxA5</i>kd cells.

<p>(A) Quantification of DNA concentration 24 hrs after cell seeding. Bars represent mean DNA concentration (ng/µL sample)±SEM, n = 5–8. (B) Quantification of Calcein-AM fluorescence 24 hrs after cell seeding. Bars represent mean calcein fluorescence units ± SEM, n = 3–8. (C) Quantification of Alamar blue absorbance 48 hrs after cell seeding. Bars represent mean optical density ± SEM, n = 8. *, **represent statistically significant difference from Si, *p<0.05, **p<0.01.</p

Effects of AnxA2 and AnxA5 knockdown on ALP and hydroxyapatite.

<p>(A) ALP activity staining in MC3T3-E1 cells (MC3T3), Si, <i>AnxA2</i>kd and <i>AnxA5</i>kd cells after culture in osteogenic differentiation media for 7, 14 and 21 days, representative images from n = 3 biological replicates. (B) Quantitation of ALP staining intensity. Bars represent mean integrated signal intensity ± SEM, n = 3. + represents statistically significant difference from same genotype on day 0, p<0.05. **represented statistically significant different from Si at the same day, p<0.01. (C) OsteoImage staining for hydroxyapatite in Si, <i>AnxA2</i>kd and <i>Anx5</i>kd cells cultured with differentiation media for 5 weeks. Each bar represents OsteoImage fluorescence normalized to PI ± SEM, n = 3. ***represents statistically significant difference from maintenance media within the same genotype, ***p<0.001; + represents statistically different from Si in same media composition, p<0.01.</p

ERK Oscillation-Dependent Gene Expression Patterns and Deregulation by Stress Response

Studies were undertaken to determine whether extracellular signal regulated kinase (ERK) oscillations regulate a unique subset of genes in human keratinocytes and subsequently whether the p38 stress response inhibits ERK oscillations. A DNA microarray identified many genes that were unique to ERK oscillations, and network reconstruction predicted an important role for the mediator complex subunit 1 (MED1) node in mediating ERK oscillation-dependent gene expression. Increased ERK-dependent phosphorylation of MED1 was observed in oscillating cells compared to nonoscillating counterparts as validation. Treatment of keratinocytes with a p38 inhibitor (SB203580) increased ERK oscillation amplitudes and MED1 and phospho-MED1 protein levels. Bromate is a probable human carcinogen that activates p38. Bromate inhibited ERK oscillations in human keratinocytes and JB6 cells and induced an increase in phospho-p38 and a decrease in phospho-MED1 protein levels. Treatment of normal rat kidney cells and primary salivary gland epithelial cells with bromate decreased phospho-MED1 levels in a reversible fashion upon treatment with p38 inhibitors (SB202190; SB203580). Our results indicate that oscillatory behavior in the ERK pathway alters homeostatic gene regulation patterns and that the cellular response to perturbation may manifest differently in oscillating vs nonoscillating cells

In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets

Nucleotides, for example, adenosine-5′-triphosphate (ATP) and guanosine-5′-triphosphate (GTP), are primary energy resources for numerous reactions in organisms including microtubule assembly, insulin secretion, ion channel regulation, and so on. In order to advance our understanding of the production and consumption of nucleoside triphosphates, a versatile sensing platform for simultaneous visualization of ATP, GTP, adenosine derivates, and guanosine derivates in living cells has been built up in the present work based on graphene oxide nanosheets (GO-nS) and DNA/RNA aptamers. Taking advantage of the robust fluorescence quenching ability, unique adsorption for single-strand DNA/RNA probes, and efficient intracellular transport capacity of GO-nS, selective and sensitive visualization of multiple nucleoside triphosphates in living cells is successfully realized with the designed aptamer/GO-nS sensing platform. Moreover, GO-nS displays good biocompatibility to living cells and high protecting ability for DNA/RNA probes from enzymatic cleavage. These results demonstrate that the aptamers/GO-nS-based sensing platform is capable of selective, simultaneous, and in situ detection of multiple nucleotides, which hold a great potential for analyzing other biomolecules in living cells

Hierarchical cluster analyses showing temporal changes in expression ratios for significant RNA and protein changes.

<p>The scale bar indicates the log₁₀ expression ratio compared to 0 hr controls. Values in gray indicate the protein/phosphorylated protein was not detected at that time point.</p

Summary of network statistics derived from individual and integrated datasets.

<p>Summary of network statistics derived from individual and integrated datasets.</p

Experimental design and characterization of cell cycle transition with EGF treatment.

<p>A) Experiments were scaled to provide sufficient sample for parallel analyses by gene microarray, global proteomics and Western blot technologies. B) Flow cytometry results showing the time course for transitions between G₁/S and G₂/M phases during EGF-induced mitosis.</p