GluCEST map of a healthy rat brain from one of the group 2 animals pre and 24 hours post Modafinil injection.

<p>(a) Anatomical brain image from one of the group 2 normal rat. (b, c) GluCEST maps show an increase in GluCEST contrast 24 hours post Modafinil administration. (d, e) Magnetization transfer ratio (MTR) maps show no observable change in MTR contrast 24 hours post Modafinil administration.</p

Chemical-exchange-saturation-transfer imaging of glutamate (GluCEST).

<p>(a) Anatomical brain image from one of the group 1 normal rat. (b) GluCEST maps of this rat brain pre and post Modafinil injection over time period of 5 hours. (c, d) B₀ and B₁ maps from corresponding brain slice as shown in (a). (e) graphs show no appreciable change in either mean GluCEST contrast or mean Glx concentration from ¹HMRS over time period of 5 hours (n = 5) for the region of interest as shown in (a).</p

Intra and inter- GluCEST coefficient of variance (CV).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103154#pone-0103154-t001" target="_blank">Table 1</a> shows the intra and inter- CV in measuring GluCEST contrast from rats brain.</p

Relative change in GluCEST and Glx concentration from ¹HMRS.

<p>Bar graphs show relative change in GluCEST contrast (a) and Glx concentration from ¹HMRS (b) 24 hours post Modafinil administration for the group 2 rats (n = 6). While the change in GluCEST was positive in all rats, the change in Glx concentration was inconsistent.</p

The antitumoral effect of SRF on human colon adenocarcinoma HCT15 xenografts.

<p>SCID female mice were implanted with 1×10⁷ HCT15 human colon adenocarcinoma cells. The treatment started when tumor size attained a 100–200 mm³ or larger. Animals were treated intraperitoneally (i.p.) with 20 mg/kg/dose SRF in final dosing formulation or a 0.9% saline vehicle. Compound was given to tumor-bearing mice on days 1, 4, and 7 after staging, and tumor mass [(length×width²)/2] was determined once a three days for 9 days. Three tumors were observed at different time points. Error bars represent standard error of the mean (n = 3). The data were analyzed by a one-sided Student’s <i>t</i> test, and values of P<0.05 were considered to be significant. *P<0.05 versus vehicle control.</p

Comparison between the effect of SRF and other microtubule binding agents on viability of drug-resistant cell types.

a<p>The KB-V1 is a mutant of the parental cell line KB-3-1 and resistant to vinblastine.</p><p>Comparison between the effect of SRF and other microtubule binding agents on viability of drug-resistant cell types.</p

Effect of SRF on BH-3 family members, Bcl-2 and Bad.

<p>(A) Western blot analysis of HeLa and WI-38 cell extracts treated with 10 µM SRF for 24 hr and probed with anti-Bcl-2 monoclonal antibody. A slower migrating band corresponding to phosphorylated form of Bcl-2 is present in HeLa extracts but absent from WI-38 lysates, showing that SRF induces selective phosphorylation of Bcl-2 in cancer cells. (B) SRF mediates Bcl-2 hyperphosphorylation. Drug treated HeLa lysates were resolved on 12% SDS-PAGE and immunoblotting was done using specific phospho-antibodies against T56, S70 and S87 residues of Bcl-2; all these amino acids lie in the flexible loop region of the protein. (C) Time course analysis of Bad phosphorylation induced by SRF treatment. HeLa cell lysates were analyzed by immunoblotting using anti-Bad monoclonal antibody. SRF (10 µM) treatment altered phosphorylation status of pro-apoptotic protein Bad as indicated by the appearance of a slower migrating phospho-Bad band at 24 h. The band was absent from control (DMSO treated) cells even after 24 h.</p

Cartoon representing plausible mechanism of SRF mediated toxicity in cancer cells.

<p>SRF can bypass P-gp mediated drug efflux via mechanism(s) that are currently unknown. Inside the cell, SRF binds and inhibits microtubule polymerization resulting in cell cycle arrest at the G₂/M phase. These events, in turn, activate JNK-mediated stress-response signaling cascade leading to the phosphorylation and inactivation of anti-apoptotic proteins like Bcl-2 and Bad. Consequently, there is loss of mitochondrial membrane potential and integrity, release of cytochrome-c, activation of caspase-3 and eventual cell death by apoptosis. Thus, SRF-mediated cell death proceeds via the intrinsic/mitochondrial apoptotic pathway.</p

Effect of SRF on viability of different cancer cell types.

<p>Effect of SRF on viability of different cancer cell types.</p

SRF cytotoxicity involves JNK kinases and proceeds via caspase-3 activation and mitochondrial membrane potential loss.

<p>(A) SRF (10 µM) activates JNK kinase but not ERK and p38. Phosphorylation status of the JNK, ERK and p38 was probed by immunoblotting using phospho-specific antibodies against the kinases. SRF selectively induces JNK phosphorylation [Panel (i)] without altering protein levels [Panel (ii)]. (B and C) Pre-treatment of cells with JNK-specific inhibitor prior to SRF (10 µM) exposure abrogates Bcl-2 and Bad phosphorylation. No phosphorylation of Bcl-2 (Panel B, Lane 4) or Bad (Panel C, Lane 3) were seen when cells were pre-treated with JNK-specific inhibitor, SP600125. Similar results were not observed with highly selective non-competitive ERK1/2 inhibitor, PD98059 (Panel B, Lane 6) or p38 inhibitor, SB203580 (Panel B, Lane 8). (D) Inhibition of JNK-kinase protects cells against SRF-induced toxicity. Cell viability was determined by MTT assay and reported as percentage control. SP600125 was able to retain viability in approximately 70% cells. Data are shown as means ± SEM. **<i>P</i><0.01 versus control. (E) Cells pre-treated with SP600125 were able to overcome SRF-induced G₂/M phase cell cycle blockage. Percentage cells in the different stages of cell cycle were determined by flow cytometric analysis. Data are shown as means ± SEM. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (F) SP600125 treated cells retain cellular microtubule network. Fluorescence micrographs of cells treated with SRF in the presence or absence of SP600125. Microtubules (green) and nucleus (blue) were stained with FITC-conjugated anti-tubulin antibody and DAPI, respectively. Scale bar = 10 µM. (G) SRF induces loss of mitochondrial membrane potential as shown by flow-cytometric analysis of cells stained with JC-1. Events were counted in the green channel. SP600125 pre-treatment prevented cells from undergoing apoptosis as percentage of cells having fluorescence in the green channel decreased from 84.2% in SRF treated cells to 47.8% for cells that were pre-treated with SP600125 prior to SRF (10 µM) exposure. (H) Apoptotic death mediated by SRF proceeds through caspase-3 activation. A cleaved band corresponding to activated caspase-3 is present in SRF-treated lysates but absent from SP600125 pre-treated lysates.</p