Percent cell death of HT-29 human colon cancer cells.

<p>At 80% confluency, cells were treated with increasing concentrations of sulindac (grey), sulindac sulfide (white), and sulindac sulfone (black).</p

Primers and probes.

1<p>All genes are human.</p

MMP7, MMP25, Trypsin1 and RPL-19 expression in sulindac sulfide-treated cells.

<p>(<b>A</b>) PCR product of RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO or sulindac sulfide. Lane 1, 50 bp DNA ladder; lane 2, <i>MMP25</i> after sulindac sulfide treatment; lane 3, <i>MMP25</i> after DMSO treatment; lane 4, <i>Trypsin1</i> after sulindac sulfide treatment; lane 5, <i>Trypsin1</i> after DMSO treatment; lane 6, <i>MMP7</i> after sulindac sulfide treatment; lane 7, <i>MMP7</i> after DMSO treatment; lane 8 <i>RPL-19</i> after sulindac sulfide treatment; lane 9, <i>RPL-19</i> after DMSO treatment. Results demonstrate that only <i>MMP7</i> expression is affected by sulindac sulfide treatment. (<b>B</b>) Quantitative RT-PCR of <i>MMP25</i>, <i>Trypsin1</i>, and <i>MMP7</i> after DMSO or sulindac sulfide treatment. The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. Results indicate quantitative differences in <i>MMP7</i> expression after sulindac sulfide treatment relative to DMSO treatment. (<b>C</b>) Western blot of secreted fraction of HT-29 cells demonstrating that the protein levels of MMP25 in the membrane fraction are similar between sulindac sulfide and DMSO treated cells.</p

Activity based proteomics and LTA4H in HT-29 cells.

<p>Metallohydrolase activity-based labeling of HT-29 cells secreted proteomes. MMP2 and MMP7 standards were added to cell proteomes (arrows). The band displaying a strong decrease (*) after sulindac sulfide treatment was extracted and analyzed by mass spectrometry. MS/MS spectra of LVVDLTDIDPDVAYSSVPYEK and LTYTAEVSVPK peptides corresponded to amino acids 366–386 and 155–165, respectively, of the LTA4H protein. Protein content across all samples was adjusted to 1 mg/ml and the equivalent of 15 µg of proteome was added per lane. MW denotes molecular weight; STDs, standards, Sd, sulindac sulfide-treated and D, DMSO-treated.</p

MMP7 expression in sulindac-treated cells.

<p>(<b>A</b>) Real time RT-PCR of RNA from HT-29 cells after 24 hours of treatment with DMSO, sulindac (S), sulindac sulfide (Sd) and sulindac sulfone (Sn). The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. C_T values between 20.10 and 20.56 across all samples show constant levels of <i>hRPL-19</i> expression. (<b>B</b>) The expression of <i>hMMP7</i> after 24 hours of sulindac treatment. The downregulation of <i>hMMP7</i> was significant at all concentrations. The results were obtained using the ΔΔCt method with <i>hRPL-19</i> as the housekeeping gene. C_T values between 23.22 and 23.56 across all samples show constant levels of <i>hRPL-19</i> expression. (<b>C</b>) Cytosolic, membrane, and secreted proteome fractions extracted from DMSO- and sulindac sulfide-treated HT-29 cells were subjected to western blot analysis using an antibody which does not differentiate between active and inactive MMP7 (top), and an antibody that only recognizes the active site (bottom). The expected band for MMP7 appears at 28 kDa, however a band at 45 kDa is often reported, possibly a dimer. Cyto denotes cytosolic fraction; Mem, membrane fraction, Secr, secreted fraction, and aMMP7, active MMP7.</p

Enzymatic immunoassay for LTB4.

<p>Secreted proteomes of DMSO-treated and sulindac sulfide-treated cells were analyzed for LTB4 levels using an enzymatic immunoassay.</p

Immunohistochemistry for Fas-L in adenomas from <i>Apc^Min/</i>⁺ and <b><i>Apc^Min/</i></b>⁺<i>/Fas^lpr</i> mice.

<p>(<b>A</b>) Immunostains for Fas-L comparing <i>Apc^Min/+/Fas^lpr</i> and <i>Apc^Min/+</i> mice at 8 and 20 weeks. (<b>B</b>) Quantitation of Fas-L immunostains. Error bars represent standard error of the mean.</p

Percentage of invasive lesions in 30 week <i>Apc^Min/+/Fas^lpr</i> mice.

<p>Percentage of invasive lesions in 30 week <i>Apc^Min/+/Fas^lpr</i> mice.</p

Conantokin-G Attenuates Detrimental Effects of NMDAR Hyperactivity in an Ischemic Rat Model of Stroke

<div><p>The neuroprotective activity of conantokin-G (con-G), a naturally occurring antagonist of N-methyl-D-aspartate receptors (NMDAR), was neurologically and histologically compared in the core and peri-infarct regions after ischemia/reperfusion brain injury in male Sprague-Dawley rats. The contralateral regions served as robust internal controls. Intrathecal injection of con-G, post-middle carotid artery occlusion (MCAO), caused a dramatic decrease in brain infarct size and swelling at 4 hr, compared to 26 hr, and significant recovery of neurological deficits was observed at 26 hr. Administration of con-G facilitated neuronal recovery in the peri-infarct regions as observed by decreased neurodegeneration and diminished calcium microdeposits at 4 hr and 26 hr. Intact Microtubule Associated Protein (MAP2) staining and neuronal cytoarchitecture was observed in the peri-infarct regions of con-G treated rats at both timepoints. Con-G restored localization of GluN1 and GluN2B subunits in the neuronal soma, but not that of GluN2A, which was perinuclear in the peri-infarct regions at 4 hr and 26 hr. This suggests that molecular targeting of the GluN2B subunit has potential for reducing detrimental consequences of ischemia. Overall, the data demonstrated that stroke-induced NMDAR excitoxicity is ameliorated by con-G-mediated repair of neurological and neuroarchitectural deficits, as well as by reconstituting neuronal localization of GluN1 and GluN2B subunits in the peri-infarct region of the stroked brain.</p></div

Immunohistochemistry for PCNA in adenomas from <i>Apc^Min/</i>⁺ and <i>Apc^Min/</i>⁺<i>/Fas^lpr</i> mice.

<p>(<b>A</b>) PCNA immunostains comparing <i>Apc^Min/+/Fas^lpr</i> to <i>Apc^Min/+</i> mice at 8 and 20 weeks. (<b>B</b>) Quantitation of PCNA positive cells per tumor area at five different time points. Error bars represent standard error of the mean.</p