Krüppel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression

<div><p>Krüppel-like factor 6 (KLF6) is a tumor suppressor gene and play a role in the regulation of cell proliferation and apoptosis. After the peripheral nerve injury (PNI), the microenvironment created by surrounding Schwann cells (SCs) is a critical determinant of its regenerative potential. In this study, we examined the effects of KLF6 on SCs responses during PNI. Both KLF6 mRNA and protein expression levels were upregulated in the injured sciatic nerve, and immunofluorescence results showed that many KLF6-positive cells simultaneously expressed the SC markers S-100 and p75NTR. The apoptosis inducers TNFα and cisplatin upregulated KLF6 expression in primary cultured SCs and the SC line RSC96. Although KLF6 overexpression exacerbated cisplatin- and TNFα-induced apoptosis, expression levels of the apoptosis regulators Bcl2 and Bax were not significantly affected in either KLF6-overexpressing or KLF6-depleted RSC96 cells. Realtime PCR arrays and qRT-PCR demonstrated that KLF6 overexpression upregulated four pro-apoptotic genes, FAS, TNF, TNFSF12, and PYCARD, and inhibited expression of the anti-apoptotic IL10 gene expression. Further analysis revealed that FAS protein expression was positively correlated with KLF6 expression in SCs. These data suggest that KLF6 upregulation may render SCs more vulnerable to apoptosis after injury via upregulating FAS expression. </p> </div

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-0

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>�l) was placed inside the three systems. The protocols are described in the Methods section. Briefly, tubes are painted black to eliminate any visual interference with exploration. One set of experiments is carried out in an aerated chamber (odour gradients are maintained), the other in grape juice-saturated atmosphere (see figure 4 and Methods section). One hundred flies were placed in the chamber and the flies inside the 3 tubes were counted 10 hours later. (A) tube with 50 μl, (B) 100 μl and (C) 300 μl. The mutants , and were analyzed along with genetic background and . Values are mean ± SEM (n = 10)

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-3

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>nts. Mutants (20 flies, 5 days old, red eyed, females to avoid pheromone interference) were released in the chamber without disturbing the aggregated spot (aggregates stable, probably because of altered vision). Simultaneously, a second fresh spot was placed inside. Here, the graph represents the time course of the ratio of aggregated flies (or ) on the pre-aggregated spot the fresh one. Values are mean ± SEM (n = 10). The mutant against control gave T value: 3.38, P = 0.003 and degrees of freedom: 18. Heat shock rescue was carried out as in figure 1. () The control of flies aggregated the total in the chamber is represented for the strains tested above. We saw no significant differences between the strains. The apparatus was the same than in the figure 5 and 6

KLF6 expression was induced in injured sciatic nerves.

<p><i>A</i>. Time course of KLF6 expression as indicated by real-time PCR following rat sciatic nerve injury. The data are normalized to GAPDH expression and presented as fold-change relative to the normal control. *<i>p</i><0.05 vs. normal control (<i>t</i>-test). <i>B</i>. Western blots showed that KLF6 expression was upregulated in a time dependent manner at the site of sciatic nerve injury, while in the sham operation group KLF6 expression showed no obvious change. <i>C</i>. and <i>D</i>. Immunohistochemical detection of KLF6, S-100 and p75NTR expression in the injured nerve. Representative images are shown and the scale bar=50 μm.</p

Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling-5

<p><b>Copyright information:</b></p><p>Taken from "Exploratory behaviour in NO-dependent cyclase mutants of shows defects in coincident neuronal signalling"</p><p>http://www.biomedcentral.com/1471-2202/8/65</p><p>BMC Neuroscience 2007;8():65-65.</p><p>Published online 6 Aug 2007</p><p>PMCID:PMC1963332.</p><p></p>hybrid GFP (first chromosome) and homozygous for mutations were analyzed for the fluorescence in the wing. The rescue in background was analyzed as double heterozygous (). Flies were heat-shocked at day 3 and analyzed two days later. Positive control: MARCM system (chromosome recombination based on flipase under heat-shock promotor induces axonal marker -)

TNFα treatment promoted KLF6 expression in SCs.

<p><i>A</i>. primary SCs were treated with 50ng/ml TNFα for time indicated and subjected to TUNEL staining. Representative images are shown along with the quantification of 5 randomly selected fields. Original magnification, 200×; *<i>p</i><0.01 vs. normal (<i>t</i>-test). <i>B</i>. Western Blotting of KLF6 expression in primary SCs or RSC96 cells (<i>C</i>) treated with TNFα.</p

KLF6 rendered SCs more susceptible to apoptotic stress.

<p><i>A</i>. KLF6 mRNA expression analyzed by real-time PCR in RSC96 cells overexpressing KLF6 (KLF6-vector) and RSC96 cells stably transfected with the empty vector (Vector). *<i>p</i><0.05 vs. Vector (<i>t</i>-test). <i>B</i>. Western blot of KLF6 expression in RSC96 cells. <i>C</i>. RSC96 cells stably transfected with control vector control or overexpressing KLF6 were treated with 100 ng/ml TNFα for 48 hours and then subjected to TUNEL staining. Representative images are shown along with the quantification of 5 randomly selected fields. Original magnification, 200×; *<i>p</i><0.05 vs. the TNF+vector group (<i>t</i>-test). <i>D</i>. RSC96 cells were treated with 16 μg/ml cisplatin for 8 hours and then subjected to TUNEL staining. Representative images are shown along with the quantification of 5 randomly selected fields. Original magnification, 200×; *p<0.05 vs. the Vector + Cisplatin group (t-test).</p

KLF6 differentially regulated apoptosis-related gene expression in SCs.

<p><i>A</i>. Real-time PCR of apoptosis-related gene expression in RSC96 cells overexpressing KLF6. *<i>p</i><0.05 <i>vs</i>. Vector (<i>t</i>-test). <i>B</i>. Real-time PCR assay of apoptosis-related gene expression in RSC96 cells with KLF6 depletion (knockout). *<i>p</i><0.05 <i>vs</i>. Negative control (NC) (<i>t</i>-test). <i>C</i> and <i>D</i>. Western blots showing FAS expression in RSC96 cells overexpressing KLF6 or with KLF6 depletion. <i>E</i>. Immunohistochemical detection of KLF6 and FAS expression in the injured nerve. Representative images are shown and the scale bar=50 μm.</p

Cisplatin treatment promoted KLF6 expression in SCs.

<p><i>A</i>. Western blotting of KLF6 expression in primary SCs treated with different doses of cisplatin. <i>B</i> and <i>C</i>. Primary SCs and the SC cell line RSC96 were treated with 16 μg/ml cisplatin and KLF6 protein expression measured at several time points. <i>D</i>. A 16 hours time course experiment on SCs treated with 16 μg/ml cisplatin, and then SCs were subjected to TUNEL staining. Representative images are shown along with the quantification of 5 randomly selected fields. Original magnification, 200×; *<i>p</i><0.01 vs. untreated cells at 0 h (<i>t</i>-test).</p

Bcl2 and Bax expression in RSC96 cells with KLF6 kncokdown or forced expression.

<p><i>A</i>. Bcl2 and Bax expression analyzed by real-time PCR in RSC96 cells with KLF6 knockdown or overexpression. <i>B</i> and <i>C</i>. Western blot of KLF6, Bcl2 and Bax expression in RSC96 cells with KLF6 knockdown or overexpression. <i>D</i>. Bax and Bcl2 expression levels were analyzed by real-time PCR. The Bax/Bcl2 ratio increased after cisplatin treatment. *<i>p</i><0.05 <i>vs</i>. untreated cells at 0 h (<i>t</i>-test). <i>E</i>. Bcl2 and Bax expression levels over 12 h as analyzed by Western blotting in RSC96 cells overexpressing KLF6. </p