MRNA expression of IFNγR was evaluated in human pancreatic carcinoid BON tumor cells (lane 3)

<p><b>Copyright information:</b></p><p>Taken from "A novel approach in the treatment of neuroendocrine gastrointestinal tumors: Additive antiproliferative effects of interferon-γ and meta-iodobenzylguanidine"</p><p>BMC Cancer 2004;4():23-23.</p><p>Published online 21 May 2004</p><p>PMCID:PMC442128.</p><p>Copyright © 2004 Höpfner et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Neuroendocrine differentiated colorectal carcinoma cells (Colo320 DM) served as positive control for IFNγR (lane 4). β-actin was used as loading control (lane 1: BON, lane 2: Colo320 DM). PCR products were visualized by gel electrophoresis on an ethidium bromide-stained agarose gel. M: 100 bp DNA ladder

Rising concentrations of IFNγ for 5 days led to a time- and dose-dependent growth inhibition of neuroendocrine gastrointestinal tumor cells, as measured by crystal violet staining

<p><b>Copyright information:</b></p><p>Taken from "A novel approach in the treatment of neuroendocrine gastrointestinal tumors: Additive antiproliferative effects of interferon-γ and meta-iodobenzylguanidine"</p><p>BMC Cancer 2004;4():23-23.</p><p>Published online 21 May 2004</p><p>PMCID:PMC442128.</p><p>Copyright © 2004 Höpfner et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> The growth of BON tumor cells was inhibited with an ICvalue of 135 ± 10 U/ml (). STC-1 cells displayed an ICvalue of about 100 U/ml (95 ± 15 U/ml) (). Data are given as percentage of untreated controls (means ± SEM of 4 experiments). *, statistical significance (< 0.05) compared to untreated controls

A combination treatment with sub-ICconcentrations of IFNγ and MIBG for 72 h led to additive growth inhibitory effects in STC-1 () and BON cells ()

<p><b>Copyright information:</b></p><p>Taken from "A novel approach in the treatment of neuroendocrine gastrointestinal tumors: Additive antiproliferative effects of interferon-γ and meta-iodobenzylguanidine"</p><p>BMC Cancer 2004;4():23-23.</p><p>Published online 21 May 2004</p><p>PMCID:PMC442128.</p><p>Copyright © 2004 Höpfner et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> Black bars indicate the values of a calculated additive growth inhibition. Data are given as percentage of untreated controls (means ± SEM of 3 experiments)

Cytotoxicity of IFNγ was evaluated by lactate dehydrogenase (LDH) release from IFNγ-treated BON cells

<p><b>Copyright information:</b></p><p>Taken from "A novel approach in the treatment of neuroendocrine gastrointestinal tumors: Additive antiproliferative effects of interferon-γ and meta-iodobenzylguanidine"</p><p>BMC Cancer 2004;4():23-23.</p><p>Published online 21 May 2004</p><p>PMCID:PMC442128.</p><p>Copyright © 2004 Höpfner et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.</p> After 48 h of incubation, IFNγ induced a dose-dependent increase in LDH release. MIBG-treatment (48 h) also induced cytotoxicity in BON cells in a dose-dependent manner. Interestingly, co-treatment of BON cells with IFNγ and MIBG resulted in an over-additive LDH release. Black bars indicate the calculated additive values of LDH release of either combination. Data are given as the relative increase in LDH release compared to untreated controls (means ± SEM of 4 independent experiments). *, statistical significance (< 0.05) compared with untreated controls

Vimentin Mediates Uptake of C3 Exoenzyme

<div><p><i>Clostridium botulinum</i> C3 exoenzyme (C3) selectively inactivates RhoA/B/C GTPases by ADP-ribosylation. Based on this substrate specificity C3 is a well-established tool in cell biology. C3 is taken up by eukaryotic cells although lacking an uptake and translocation domain. Based on different approaches vimentin was identified as membranous C3-interaction partner by mass spectrometry. Vimentin in fact was partly localized at the outer surface of hippocampal HT22 cells and J744A.1 macrophages. Domain analysis identified the rod domain as binding partner of C3. Vimentin was also involved in uptake of C3 as shown by knock down of vimentin in HT22 and J774A.1 cells. The involvement of vimentin in uptake of C3 was further supported by the findings that the vimentin disruptor acrylamide blocked uptake of C3. Vimentin is not only a major organizing element of the intermediate filament network but is also involved in both binding and uptake of C3 exoenzyme.</p></div

Uptake of C3 in HT22 and J744A.1 cells is dependent on vimentin distribution and integrity.

<p>A) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into HT22 cells detected as RhoA degradation (induced by C3-catalysed ADP-ribosylation). In a pulse-chase experiment, HT22 cells were incubated with C3 (500 nM) at 4°C for 60 min. Afterwards unbound C3 was removed by washing the cells three times with PBS and fresh medium was added. Cells were then cultivated for further 48 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). B) Cellular levels of RhoA proteins were quantified by densitometric evaluation of RhoA (from A) and adjusted to the corresponding actin band. C) HT22 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 24 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin. C3 alone causes a complete mol weight shift of RhoA in SDS-PAGE. Western blot analysis of one representative experiment is shown (n = 3 independent experiments). D) RhoA shift (indicative of Rho-ADP-ribosylation) by quantified by densitometric evaluation of RhoA (from C) and adjusted to the corresponding β-actin signal. E) Influence of Vim-siRNA knock down (for 48 h) on the uptake of C3 into J774A.1 cells detected as incomplete RhoA ADP-ribosylation. J774A.1 macrophages were incubated with C3 (500 nM) at 37°C for 4 h. Cell lysates were generated and separated by SDS-PAGE followed by Western blot analysis probing RhoA and β-actin. One representative experiment is shown (n = 3 independent experiments). F) J774A.1 cells were pre-treated with acrylamide (5 mM) for 30 min followed by incubation with C3 (500 nM) for 4 h. Cells were lysed and submitted to Western blot analysis probing RhoA and β-actin.</p

Binding of C3 to HT22 cells after pronase treatment.

<p>A) Pronase pre-incubated HT22 cells were exposed to 100 or 500 nM of C3 for 1 h at 4°C. Subsequently, β-actin and bound C3 were detected by Western blot. NC = negative control without C3, PC = positive control lysate with 10 ng C3. One representative experiment is shown (n = 3 independent experiments). B) Pronase-treated HT22 cells were exposed to 500 nM of C3-E174Q-FITC for 1 h at 4°C and bound C3- E174Q-FITC was analyzed by FACS.</p