Journal: Cell metabolism

Article Title: Systematic dissection of the metabolic-apoptotic interface in AML reveals heme biosynthesis to be a regulator of drug sensitivity

doi: 10.1016/j.cmet.2019.01.011

Figure Lengend Snippet:

Article Snippet: Cat# ab14745, RRID:AB_2213640; Rabbit polyclonal anti-Bax (N-terminus) EMD Millipore Cat# 06–499, RRID:AB_310143 Rabbit monoclonal anti-Bcl-2 (clone D55G8) antibody Cell Signaling Technology Cat# 4223, RRID:AB_1903909 Rabbit monoclonal anti-Bcl-xL (clone 54H6) antibody Cell Signaling Technology Cat# 2764, RRID:AB_2228008 Rabbit monoclonal anti-Mcl-1 (clone S-19) antibody Santa Cruz Biotechnology Cat# sc-819, RRID:AB_2144105 Rabbit monoclonal anti-Bim (clone C34C5) antibody Cell Signaling Technology Cat# 2933, RRID:AB_1030947 Rabbit polyclonal anti-Bid antibody Cell Signaling Technology Cat# 2002, RRID:AB_10692485 Rabbit monoclonal anti-total Bad (clone D24A9) antibody Cell Signaling Technology Cat# 9239, RRID:AB_2062127 Rabbit monoclonal anti-phospho-Bad (S112) (clone 40A9) antibody Cell Signaling Technology Cat# 5284, RRID:AB_560884 Rabbit monoclonal anti-phospho-Bad (S136) (clone D25H8) antibody Cell Signaling Technology Cat# 4366, RRID:AB_10547878 Rabbit monoclonal anti-PUMA (clone D30C10) antibody Cell Signaling Technology Cat# 12450 Rabbit monoclonal anti-NOXA (clone D8L7U) antibody Cell Signaling Technology Cat# 14766 Bacterial and Virus Strains lentiCRISPR v2 Sanjana et al. Nat.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, Cell Fractionation, Membrane, Cell Culture, Cell Viability Assay, Activity Assay, Isolation, shRNA, Metabolic Labelling, Software

Journal: Cell metabolism

Article Title: Mitofusin 2 regulates axonal transport of calpastatin to prevent neuromuscular synaptic elimination in skeletal muscles

doi: 10.1016/j.cmet.2018.06.011

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Article Snippet: Rabbit polyclonal anti-Bim , Cell Signaling Technology , Cat# 2819S, RRID: AB_10692515.

Techniques: Histone Deacetylase Assay, Virus, Plasmid Preparation, Control, Recombinant, Ointment, Lysis, Western Blot, Transfection, Protease Inhibitor, Sample Prep, Transgenic Assay, Software

Journal: Cell Division

Article Title: M2I-1 disrupts the in vivo interaction between CDC20 and MAD2 and increases the sensitivities of cancer cell lines to anti-mitotic drugs via MCL-1s

doi: 10.1186/s13008-019-0049-5

Figure Lengend Snippet: The majority of cell deaths occurred after prolonged mitotic arrest. a Cell extracts were prepared from HeLa cells after 16 h of the different drug treatments as described previously. The western blot membranes were probed with a rabbit polyclonal anti-phospho-histone H3 (S-10) antibody (Millipore, #06-570) (1:500 dilutions), and the actin protein bands acted as the loading control. b Quantitative comparison of the phospho-histone 3 S-10 bands. Results produced from three independent experiments. c The confocal time-lapse images showing an example of a normal/unperturbed HeLa H2B-GFP cell undergoing the cell cycle in mitosis. d An example of a HeLa H2B-GFP cell undergoing apoptosis after prolonged mitotic arrest by 60 ng/ml nocodazole or 60 ng/ml nocodazole + 50 μM M2I-1. The white arrowhead indicates the time point when the cytoplasmic membrane (DIC grey images) of the cell starts shrinking and rounding up with condensed chromosomes (green). The diamond heads indicate the time point that the cytoplasmic membrane began blebbing. The chromosomes displaying fragmentation and over-condensation are highlighted by white dashed arrows and normal arrows respectively. A typical apoptotic-body like morphology is highlighted with the asterisk. e An example of a HeLa H2B-GFP cell undergoing apoptosis at a prophase-like stage induced by treatment with 60 ng/ml nocodazole + 50 μM M2I-1. The white arrowhead highlights the time point where there is clear cell body shrinkage, dash arrows highlight the fragmenting chromosomes and normal black arrows indicate the intact cytoplasmic membrane during chromosome fragmentation. Asterisks highlight the formation of a typical apoptotic-body like morphology. The green histone 2B-GFP highlights the chromosomal morphologies and the DIC images in grey indicate the cellular boundaries and the cytoplasmic membrane. The timing of the images is indicated

Article Snippet: Primary antibodies used in this project are Rabbit polyclonal anti-CDC20 antibody (Abcam, ab26483); mouse monoclonal anti-p55 CDC (E-7) (Santa Cruz Biotech, sc-13162); rabbit polyclonal anti-full length MAD2 (Convance, PRB-452C); mouse monoclonal anti-cyclin B1 (GNS) (Santa Cruz, sc-245); mouse monoclonal anti-cyclin A (B-8) (Santa Cruz, sc-271682); mouse monoclonal anti-actin antibody (Abcam, ab6276); mouse monoclonal anti-GADPH antibody (Thermo Fisher Scientific, MA5-15738); rabbit polyclonal anti-caspase-3 antibody (Abcam, ab32351); rabbit polyclonal anti-phospho-histone 3 (S-10) antibody (Millipore, #06-570); and rabbit polyclonal anti-GFP antibody [Santa Cruz, sc-8334 (GP-FL)]; rabbit polyclonal anti-γ-H2AX (S-139) antibody (Abcam, ab-2893); GFP-Trap A geta-20 (ChromoTek, 70112001A); rabbit polyclonal anti-Mcl-1 (S-19) antibody (Santa Cruz, sc-819); rabbit polyclonal anti-pericentrin 1&2 antibody (Abcam, ab4448); rabbit polyclonal anti-BID (FL-195) antibody (Santa Cruz, sc-11423); mouse monoclonal anti-BIM (H-5) antibody (Santa Cruz, sc-374358); rabbit polyclonal anti-NOXA (FL-54) antibody (Santa Cruz, sc-30209) and monoclonal anti-PUMA (G-3) antibody (Santa Cruz, sc-374223); rabbit monoclonal anti-human PARP-1 antibody (46D11) (Cell Signaling Technology).

Techniques: Western Blot, Control, Comparison, Produced, Membrane

Journal: Cell Division

Article Title: M2I-1 disrupts the in vivo interaction between CDC20 and MAD2 and increases the sensitivities of cancer cell lines to anti-mitotic drugs via MCL-1s

doi: 10.1186/s13008-019-0049-5

Figure Lengend Snippet: The regulation of the stability of MCL-1, cyclin B1, and other pro- or anti-apoptotic proteins in HeLa and MCF-7 cells. a Western blots of cell extracts prepared from HeLa cells after treatment with 0.5% DMSO, 50 μM M2I-1, 60 ng/ml nocodazole, and 60 ng/ml nocodazole + 50 μM M2I-1 respectively and probed with a rabbit polyclonal anti-cyclin B1 antibody (H-433) antibody (sc-7520); a mouse monoclonal anti-actin (AC-15) antibody (ab6276) was used as the loading control. b – f Western blot results showing the expression levels of the pro-apoptotic proteins BIM, BID, PUMA, NOXA, and MCL-1s in HeLa cells. Cell extracts were prepared from HeLa cells after treatment with nocodazole alone or nocodazole + M2I-1 as described before. Actin bands were used as the loading controls. MCF-7 cells were treated using the same conditions as described in Fig. e apart from the determination of the chromosomal morphologies, which was highlighted by staining with 0.2 μ M SiR-DNA. Digital images were taken after 20 h incubation for quantification of the mitotic ( g ), apoptotic ( h ), and slippage indices ( i ). n: The number of the parallel experiments and the total cell numbers used for quantification. P value * < 0.026, 0.029, and 0.036 respectively

Article Snippet: Primary antibodies used in this project are Rabbit polyclonal anti-CDC20 antibody (Abcam, ab26483); mouse monoclonal anti-p55 CDC (E-7) (Santa Cruz Biotech, sc-13162); rabbit polyclonal anti-full length MAD2 (Convance, PRB-452C); mouse monoclonal anti-cyclin B1 (GNS) (Santa Cruz, sc-245); mouse monoclonal anti-cyclin A (B-8) (Santa Cruz, sc-271682); mouse monoclonal anti-actin antibody (Abcam, ab6276); mouse monoclonal anti-GADPH antibody (Thermo Fisher Scientific, MA5-15738); rabbit polyclonal anti-caspase-3 antibody (Abcam, ab32351); rabbit polyclonal anti-phospho-histone 3 (S-10) antibody (Millipore, #06-570); and rabbit polyclonal anti-GFP antibody [Santa Cruz, sc-8334 (GP-FL)]; rabbit polyclonal anti-γ-H2AX (S-139) antibody (Abcam, ab-2893); GFP-Trap A geta-20 (ChromoTek, 70112001A); rabbit polyclonal anti-Mcl-1 (S-19) antibody (Santa Cruz, sc-819); rabbit polyclonal anti-pericentrin 1&2 antibody (Abcam, ab4448); rabbit polyclonal anti-BID (FL-195) antibody (Santa Cruz, sc-11423); mouse monoclonal anti-BIM (H-5) antibody (Santa Cruz, sc-374358); rabbit polyclonal anti-NOXA (FL-54) antibody (Santa Cruz, sc-30209) and monoclonal anti-PUMA (G-3) antibody (Santa Cruz, sc-374223); rabbit monoclonal anti-human PARP-1 antibody (46D11) (Cell Signaling Technology).

Techniques: Western Blot, Control, Expressing, Staining, Incubation

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