Journal: Molecular oncology

Article Title: miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours.

doi: 10.1016/j.molonc.2015.03.013

Figure Lengend Snippet: Figure 4 e miR-221 and miR-222 dependent induction of apoptosis is mediated by the KIT signalling cascade in the imatinib sensitive cell line GIST882. Cells were treated for 24, 48 and 72 h with miR-221, miR-222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock). A) MiR-221 and miR-222 mediate reduced expression of phosphorylated and total KIT protein in the cell line GIST882. B) Western blot analyses of total KIT and phosphorylated KIT after RNA interference showing that the expression of both proteins markedly decreased after transfection of KIT siRNA compared to non-targeting AllStars negative control siRNA (N.C.) and Mock control C) Analyses of the downstream signalling cascade of KIT after transfection of miRNAs revealed a decrease in phosphorylated and total AKT and total BCL2. Phosphorylated and total MTOR as well as total BCL2L11 are less affected.

Article Snippet: The membranes were then probed with specific primary antibodies and incubated at 4 C overnight: p-AKT (1:500, Ser473, monoclonal rabbit anti p-AKT), AKT (1:500, polyclonal rabbit anti AKT), p-MTOR (1:500, Ser2448, polyclonal rabbit anti p-mTOR), BCL2 (1:1000, polyclonal rabbit anti BCL2) and ACTB (also known as beta-actin; 1:500, monoclonal mouse anti ACTB, all purchased from Cell Signaling Technology , Danvers, US), BCL2L11 (1:250, polyclonal rabbit anti BCL2L11) and MTOR (1:500, polyclonal rabbit anti MTOR, both purchased from Acris Antibodies GmbH, Herford, DE).

Techniques: Concentration Assay, Transfection, Expressing, Western Blot, Negative Control, Control

Journal: Molecular oncology

Article Title: miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours.

doi: 10.1016/j.molonc.2015.03.013

Figure Lengend Snippet: Figure 5 e miR-221 and miR-222 dependent induction of apoptosis is mediated by the KIT signalling cascade in the imatinib sensitive cell line GIST-T1. Western blot analyses of GIST-T1 cells treated with miR-221, miR-222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock) at three different time points (24, 48 and 72 h). A) MiR-221 and miR-222 reduced the expression of phosphorylated and to a lesser extend total KIT protein expression in the cell line GIST-T1 shown by Western blot. B) RNA interference abolished almost completely the expression of phosphorylated and total KIT protein in the cell line GIST-T1. C) Western blot analyses showing a reduction of phosphorylated AKT, total AKT and to a lesser extent total BCL2 expression after transfection of miR-221, miR-222 and a combination of both in the cell line GIST-T1. miRNA transfection had not much influence on phosphorylated and total MTOR as well as on total BCL2L11.

Article Snippet: The membranes were then probed with specific primary antibodies and incubated at 4 C overnight: p-AKT (1:500, Ser473, monoclonal rabbit anti p-AKT), AKT (1:500, polyclonal rabbit anti AKT), p-MTOR (1:500, Ser2448, polyclonal rabbit anti p-mTOR), BCL2 (1:1000, polyclonal rabbit anti BCL2) and ACTB (also known as beta-actin; 1:500, monoclonal mouse anti ACTB, all purchased from Cell Signaling Technology , Danvers, US), BCL2L11 (1:250, polyclonal rabbit anti BCL2L11) and MTOR (1:500, polyclonal rabbit anti MTOR, both purchased from Acris Antibodies GmbH, Herford, DE).

Techniques: Western Blot, Concentration Assay, Transfection, Expressing

Journal: Molecular oncology

Article Title: miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours.

doi: 10.1016/j.molonc.2015.03.013

Figure Lengend Snippet: Figure 6 e miR-221 and miR-222 dependent induction of apoptosis is mediated by the KIT signalling cascade even in the imatinib resistant cell line GIST48. Effects of the transfection of miR-221, miR-222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock) was measured by Western blot in the cell line GIST48 at three different time points. A) Reduction of phosphorylated KIT could be observed after 48 h whereas total KIT was already reduced after 24 h of transfection. B) Western blot after RNA interference showing a strong reduction of phosphorylated as well as total KIT protein expression already after 24 h. C) Induction of apoptosis is mediated by a downregulation of phosphorylated and total AKT as well as total BCL2 protein expression but not by regulation of phosphorylated and total MTOR protein or total BCL2L11 in the cell line GIST48.

Article Snippet: The membranes were then probed with specific primary antibodies and incubated at 4 C overnight: p-AKT (1:500, Ser473, monoclonal rabbit anti p-AKT), AKT (1:500, polyclonal rabbit anti AKT), p-MTOR (1:500, Ser2448, polyclonal rabbit anti p-mTOR), BCL2 (1:1000, polyclonal rabbit anti BCL2) and ACTB (also known as beta-actin; 1:500, monoclonal mouse anti ACTB, all purchased from Cell Signaling Technology , Danvers, US), BCL2L11 (1:250, polyclonal rabbit anti BCL2L11) and MTOR (1:500, polyclonal rabbit anti MTOR, both purchased from Acris Antibodies GmbH, Herford, DE).

Techniques: Transfection, Concentration Assay, Western Blot, Expressing