Journal: Cancer research

Article Title: BCL-2 IS A THERAPEUTIC TARGET FOR HYPODIPLOID B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

doi: 10.1158/0008-5472.CAN-18-0236

Figure Lengend Snippet: A) Leukemic cells collected from the spleens of mice at the end of the trial were analyzed for levels of the prosurvival proteins: Bcl-2, Bcl-xL, MCL-1, and the proapoptotic BIM (EL (extra long), L (long) and S (short) isoforms), BAD, PUMA and BAX. Left panel corresponds to the control B-ALL: Ph+ and Ph-Like ALL while middle and right panels are LH and NH, respectively. Each lane represents individually xenografted mice: vehicle treated (V) and ABT-199 treated (ABT). Numbers correspond to the numbers given to the mice engrafted with each leukemia (1–6). Lane numbers highlighted in purple indicate mice with high IC50 to ABT-199 at end of trial. Solid line boxes highlight the mice with predominance of BIM and the dotted line boxes highlight mice with absence of BIM and predominance of BAD and PUMA. B) NALM-16 and Beck-1732 were exposed to increasing concentrations of ABT-199 for 24h and levels of the indicated proteins measured by WB. C) Beck-1732 cells were exposed to increasing doses of ABT-199 for 24 and 48h and IC50 levels determined.

Article Snippet: ATM (CST, 2873S), Actin-HRP (CST, 5125), BAD (Abcam, ab32445), BAX (Bio-Rad, MCA2738), Bcl-2 (Abcam, ab32124), Bcl-xL (Abcam, 32370), Bcl-w (LSBio, LS-C382259), BIM (Bio-Rad, AHP933), BMF (Abcam, ab 181148), BIK (CST 4592S), BAK (Abcam, ab32371), BID (CST, 2002T), Cleaved Caspase-3, (CSST, 9664L) c-CBL (CST, 2747), pCDK2 (Abcam, ab76146), pERK (T202/Y204) (CST, 9101), Cleaved-PARP (Abcam, ab32064), PTEN (Abcam, ab32199), PUMA (LifeSpan Biosciences, LS- {"type":"entrez-nucleotide","attrs":{"text":"C98860","term_id":"3761612","term_text":"C98860"}} C98860 –400), PARP (Abcam, ab32138), HRK (BIO-RAD AHP1178T), MCL-1 (Abcam, ab32087), pRaf1 (S338) (CST, 9424), pS6 (S235/236) (CST, 2211), pSTAT5 (Y694) (BD, 611964), pmTOR (S2448) (CST, 2976), pTyr (CST, 9411), p14 (CST, 2407S), p16 (Biolegend, 675602), p21 (CST 2947S), p27 (CST, 3688T), p53 (Bio-Rad, MCA1701), p63 (ProteinTech, 12143–1AP), p73 (Abcam, ab197040).

Techniques: Control

Journal: Cancer research

Article Title: BCL-2 IS A THERAPEUTIC TARGET FOR HYPODIPLOID B-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

doi: 10.1158/0008-5472.CAN-18-0236

Figure Lengend Snippet: A) Different leukemia cell lines from different hematopoietic lineages were subjected to 100 nM of ABT-199 for 24h. Proliferation was measured using ATP-bioluminescence (Cell Titer Glo) and %Max proliferation is shown. B) Bcl-2, Bcl-xL and BAX protein levels from different leukemia cell lines are shown. Vertical line has been inserted to indicate a repositioned gel lane. C) Levels of Bcl-2, Bcl-xL, MCL-1, BAX, BIM, BAD and PUMA proteins were measured in two near haploid cell lines (NALM-16, and MHH-CALL2). CCRF-CEM was used as negative control (low Bcl-2) and HL60 as positive control (high Bcl-2 levels) for sensitivity to ABT-199.

Article Snippet: ATM (CST, 2873S), Actin-HRP (CST, 5125), BAD (Abcam, ab32445), BAX (Bio-Rad, MCA2738), Bcl-2 (Abcam, ab32124), Bcl-xL (Abcam, 32370), Bcl-w (LSBio, LS-C382259), BIM (Bio-Rad, AHP933), BMF (Abcam, ab 181148), BIK (CST 4592S), BAK (Abcam, ab32371), BID (CST, 2002T), Cleaved Caspase-3, (CSST, 9664L) c-CBL (CST, 2747), pCDK2 (Abcam, ab76146), pERK (T202/Y204) (CST, 9101), Cleaved-PARP (Abcam, ab32064), PTEN (Abcam, ab32199), PUMA (LifeSpan Biosciences, LS- {"type":"entrez-nucleotide","attrs":{"text":"C98860","term_id":"3761612","term_text":"C98860"}} C98860 –400), PARP (Abcam, ab32138), HRK (BIO-RAD AHP1178T), MCL-1 (Abcam, ab32087), pRaf1 (S338) (CST, 9424), pS6 (S235/236) (CST, 2211), pSTAT5 (Y694) (BD, 611964), pmTOR (S2448) (CST, 2976), pTyr (CST, 9411), p14 (CST, 2407S), p16 (Biolegend, 675602), p21 (CST 2947S), p27 (CST, 3688T), p53 (Bio-Rad, MCA1701), p63 (ProteinTech, 12143–1AP), p73 (Abcam, ab197040).

Techniques: Negative Control, Positive 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: 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

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: 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: 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