25.当院における内視鏡的乳頭切開術の臨床的検討(第617回千葉医学会例会・第1内科教室同門会例会)

Primer sequences and amplicon sizes. (DOCX 15 kb

Inhibition of late stage autophagy by TQ.

<p>Pathway shows initiation of autophagosome formation, however, LC3-II and p62 accumulate due to TQ-mediated decrease in autophagosome degradation. TQ also mediates lysosome permeability resulting in leakage of cathepsin B into the cytosol where it can signal to downstream mediators of cell death.</p

Chloroquine inhibits glioblastoma cell proliferation.

<p>U87MG and Gli36ΔEGFR cells were incubated with increasing concentrations of CQ for 7–10 days. At the end of the growth period colonies were fixed, stained and counted. After taking into account the plating efficiency for each cell line, the surviving fraction is the ratio of the number of colonies counted and the initial number of cells plated. Mean ± S.D., n = 3.</p

Caps-2S inhibits cisplatin-induced membrane blebbing.

<p>A, CDDP cells were transfected with casp-2S siRNA or control siRNA for 48 h, and treated with cisplatin for 48 h. The apoptotic bleb and dead cells were stained separately as described in Materials and Methods. B, The number of apoptotic blebbing cells were counted and plotted. n = 50, bar: SD, *: p<0.05 compared to siControl.</p

TQ inhibits glioblastoma cell proliferation.

<p>A) T98G, U87MG and Gli36ΔEGFR cells were incubated with increasing concentrations of TQ for 7–10 days. At the end of the growth period colonies were fixed, stained and counted. After taking into account the plating efficiency for each cell line, the surviving fraction is the ratio of the number of colonies counted and the initial number of cells plated. Mean ± S.D., n = 3. B) The effect of TQ on proliferation of normal human astrocytes (NHAs) compared to Gli36ΔEGFR cells was determined by a modified clonogenic assay. Cells were plated at a low density and incubated with increasing concentrations of TQ for 5 days. At the end of the growth period, cells were fixed, stained and photographed for estimation of proliferation relative to untreated cells.</p

Casp-2S interacts with cytoskeleton proteins.

<p>A, Casp-2S was purified from HeLa cells stably transfected with the FLAG-tagged casp-2S using an anti-FLAG affinity gel. Purified proteins were separated by SDS-PAGE and stained with Coomassie blue. The differentially expressed bands in the HeLa-casp-2S cells were identified by mass-spectrometry. B, FLAG-tagged casp-2S was immunoprecipitated from the HeLa-Casp-2S cells and Fodrin was detected using Western blotting with the anti-Fodrin antibody. C, D, Fodrin was immunoprecipitated from the HeLa-Casp-2S cells (C) or CDDP cells (D) with an anti-Fodrin antibody, and the existence of casp-2S was detected with anti-casp-2S antibody.</p

Casp-2S and cleavage of Fodrin mainly occur in cytoplasm.

<p>A, HeLa cells were UV irradiated at 20 J/m², and further cultured for 4 h. Cytoplasm (C) and nuclei (N) were separated and the Fodrin cleavage in protein extracts was detected with an anti-Fodrin antibody. Tubulin and PARP were used to indicate the cytoplasm and nuclei fraction, respectively. B. CDDP cells were fractionated into cytoplasm and nuclei, the endogenous casp-2L and casp-2S were detected simultaneously with the anti-casp-2 antibody. C, Two clones of casp-2S stably expressed HeLa cells were fractionated to the cytoplasm and nuclei, casp-2L and FLAG-tagged casp-2S were detected with the anti-casp-2 and the anti-FLAG antibodies, respectively. D, HCT116 p53^−/− and HCT116 p53^+/+ cells were transiently transfected with FLAG-tagged casp-2S, UV irradiated at various doses, and further cultured for 4 h. The cytoplasm and nuclei were separated, the Fodrin cleavage, casp-2L, and FLAG-tagged casp-2S were detected with anti-Fodrin, anti-casp-2 and anti-FLAG antibodies, respectively.</p

TQ blocks autophagy in glioblastoma cells.

<p>U87MG and Gli36ΔEGFR cells were treated with increasing concentrations of TQ, with and without chloroquine (40 µM), harvested at 24 hours and immunoblotted for A) microtubule-associated light chain (LC3-I and LC3-II), and B) p62. C) Cells were treated with TQ, with and without chloroquine, harvested at 1 hour and immunoblotted for Beclin-1. LC3-II, p62 and Beclin-1 levels were normalized to actin. Graphs show protein levels relative to untreated control.</p

Lysosomal membrane permeabilization (LMP) and cathepsin activity is involved in thymoquinone-induced cell death.

<p>A) Cytoplasmic vacuolization was observed in U87MG cells after treatment for 1 hour with either TQ or chloroquine, but not in Gli36ΔEGFR cells. At 6 hours, vacuolization can be seen in both cell types. B) Treatment with TQ or chloroquine for 6 hours induces lysosome membrane permeability in U87MG and Gli36ΔEGFR cells, seen as the loss of red acridine orange staining. C) Cells were pre-treated with cathepsin inhibitor III, which primarily targets cathepsin B, for 1 hour. TQ was added and incubated with the cells for 24 hours. Cell viability was measured using the MTT assay.</p

Casp-2S inhibits cisplatin-induced phosphatidylserine externalization.

<p>Casp-2S was over-expressed in A2780 cells (A), or knocked down in CDDP cells (B) for 48 h. Cells were then treated with cisplatin for another 24 and 48 h, and the phosphatidylserine externalization was detected with Annexin V staining by using Flow cytometry. n = 3, bar: SD, *: p<0.05 compared to the cisplatin-treated vector or control siRNA transfected cells at the same time point.</p