Journal: Cell Death & Disease

Article Title: BCL-xL/BCL2L1 is a critical anti-apoptotic protein that promotes the survival of differentiating pancreatic cells from human pluripotent stem cells

doi: 10.1038/s41419-020-2589-7

Figure Lengend Snippet:

Article Snippet: Anti-MCL1 (Rabbit polyclonal) , Cell Signaling Technology , #4572; RRID: AB_2281980.

Techniques: Virus, shRNA, Recombinant, Saline, Knock-Out, Transfection, Protein Extraction, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Purification, Isolation, Software, Spectrophotometry

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) Endogenous MCL1 is targeted by acetylation in HeLa cells. Shown is immunoblot (IB) analysis of whole-cell lysates (WCLs) and anti-MCL1 immunoprecipitates derived from HeLa cells. (B) p300 promotes MCL1 acetylation in cells. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with Myc-MCL1 along with the indicated hemagglutinin (HA)-tagged lysine acetyltransferase constructs. (C and D) Endogenous interaction between MCL1 and p300. Shown is IB analysis of WCLs (input) and anti-p300 immunoprecipitates (C) or anti-MCL1 immunoprecipitates (D) derived from HeLa cells. (E) Knockdown of p300 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the lentiviral short hairpin RNA (shRNA) specific for GFP or p300 . Cells were treated with MG132 (10 μM) for 6 h before harvesting to stabilize and accumulate MCL1 protein. (F) Treatment with the p300/CBP inhibitor A-485 reduces MCL1 acetylation. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells. Cells were co-treated overnight with A-485 at the indicated concentration and MG132 (5 μM) before harvesting. (G) Acetylation-deficient K40R markedly diminishes p300-mediated MCL1 acetylation. Shown is IB analysis of WCLs and anti-Myc tag immunoprecipitates derived from 293T cells transfected with the indicated Myc-MCL1 constructs. (H) K40 of endogenous MCL1 is targeted by acetylation in SKBR3 breast cancer cells. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from SKBR3 cells. (I) MCL1 and p300 are present together in cytoplasmic and nuclear compartments. Shown is IB analysis of WCLs and cytoplasmic, mitochondrial, and nuclear fractions derived from BT-20 cells. Data are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Western Blot, Derivative Assay, Transfection, Construct, Knockdown, Stable Transfection, Expressing, shRNA, Concentration Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) p300 knockdown decreases MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells infected with the indicated lentiviral shRNA specific for GFP or p300 . (B) p300 knockdown shortens MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the indicated lentiviral shRNA. The cells were treated with the protein synthesis inhibitor cycloheximide (CHX; 100 μg/mL) for the indicated periods before harvesting. (C) Quantification of the MCL1 band intensities of IB replicates in (B). Data are presented as mean ± SD; n = 3 independent experiments, *p < 0.05. (D) Treatment with the p300/CBP inhibitor A-485 shortens MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells. Cells were pretreated with A-485 (3 μM) overnight and then treated with 100 μg/mL CHX for the indicated periods before harvesting. (E) Quantification of the MCL1 band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01. (F) HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or p300 were transfected with His-tagged ubiquitin (His-Ub) and Myc-MCL1. 36 h after transfection, the cells were treated overnight with MG132 (10 μM) before harvesting. His-Ub-conjugated proteins were captured with Ni(2+)-nitrilotriacetic acid (Ni-NTA) agarose beads and subjected to IB analysis. (G) Acetylation-mimetic K40Q substitution results in decreased poly-ubiquitination of MCL1 in cells. 293T cells were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated overnight with MG132 (10 μM) before harvesting. (H) Ectopic p300 expression decreases poly-ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with the indicated constructs. 36 h after transfection, cells were treated with MG132 (20 μM) for 5 h and harvested for the Ni-NTA pull-down. (I) Treatment with the p300 inhibitor A-485 induces poly-ubiquitination of the WT but not the K40R mutant form of MCL1. 293T cells were transfected with the indicated constructs. 24 h after transfection, cells were treated with or without A-485 (3 μM) for 24 h and MG132 (10 μM) for 10 h before harvesting. (J) Acetylation-mimetic K40Q extends MCL1 protein half-life. Shown is IB analysis of WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 constructs. 48 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting. (K) Quantification of the Myc band intensities of IB replicates in (J). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01. (L) Ectopic p300 expression extends the protein half-life of the WT but not the K40R form of MCL1. Shown is IB analysis of WCLs derived from 293T cells transfected with Myc-MCL1 and HA-p300 constructs as indicated. 48 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting. (M) Quantification of the Myc band intensities of IB replicates in (L). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01; NS, not significant. Data in (A) and (F)–(I) are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Knockdown, Quantitative Proteomics, Derivative Assay, Infection, shRNA, Stable Transfection, Expressing, Transfection, Ubiquitin Proteomics, Construct, Mutagenesis

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A and B) Ectopic expression of the acetylation-mimetic MCL1 K40Q mutant confers resistance to doxorubicin-induced downregulation. Shown is IB analysis of WCLs derived from HeLa cells (A) and HCT116 cells (B), which stably express WT MCL1, K40Q, or K40R at a level comparable to that where endogenous MCL1 is eliminated by the CRISPR-Cas9 system. The resulting cells were treated with doxorubicin (1 μg/mL) for 24 h before harvesting. (C) Acetylation-mimetic MCL1 K40Q enhances its anti-apoptotic function. The HCT116 cells presented in (B) were treated with the indicated concentrations of doxorubicin for 24 h and then subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates; **p < 0.01, ***p < 0.001. (D and E) Acetylation-mimetic MCL1 K40Q enhances the tumorigenic activity of MCL1. A colony formation assay was conducted using HeLa cells (D) presented in (A) and a soft agar assay using HCT116 cells (E) presented in (B). These cells were pretreated overnight with doxorubicin (0.02 μ g/mL) before plating for the assays. Data are presented as mean ± SD; n = 3 biological replicates; *p < 0.05, ***p < 0.001. (F–H) Acetylation-mimetic MCL1 K40Q promotes tumor growth in the mouse xenograft model. HeLa cells presented in (A) were injected subcutaneously into nude mice (n = 6 for each group). Tumor growth was monitored over the indicated periods (F) and the weight of the dissected tumors (G) and images of the dissected tumors (H) are presented. Data are presented as mean ± SEM; n = 6; *p < 0.05, **p < 0.01, ***p < 0.001. Data in (A) and (B) are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Expressing, Mutagenesis, Derivative Assay, Stable Transfection, CRISPR, Activity Assay, Colony Assay, Soft Agar Assay, Injection

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) Acetylation-mimetic MCL1 K40Q enhances the interaction between MCL1 and USP9X. Shown is IB analysis of WCLs and anti-FLAG immunoprecipitates derived from 293T cells transfected with the indicated constructs. (B) USP9X depletion reverses the ubiquitination levels of acetylation-mimetic MCL1 K40Q. HeLa cells stably expressing the indicated lentiviral shRNA were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated overnight with MG132 (10 μM) before harvesting. (C) Treatment with the USP9X inhibitor reverses ubiquitination of acetylation-mimetic MCL1 K40Q. HeLa cells were transfected with the indicated Myc-MCL1 and His-Ub constructs. 36 h after transfection, the cells were treated with MG132 (10 μM) in the presence or absence of WP1130 (5 μM) for 4 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: quantification of the Myc-poly-ubiquitination band intensities of IB replicates. Data are presented as mean ± SD, n = 3 independent experiments, *p < 0.05, **p < 0.01. (D) USP9X depletion abolishes stabilization of MCL1 K40Q. HeLa cells stably expressing the lentiviral shRNA specific for GFP or USP9X were transfected with the indicated Myc-MCL1 constructs. 36 h after transfection, cells were treated with 100 μg/mL CHX for the indicated periods before harvesting. (E) Quantification of the Myc band intensities of IB replicates in (D). Data are presented as mean ± SD; n = 3 independent experiments, **p < 0.01. (F and G) Treatment with the USP9X inhibitor WP1130 efficiently downregulates acetylation-mimetic MCL1 K40Q. Shown is IB analysis of WCLs derived from the MCL1-reintroduced CRISPR-Cas9-mediated MCL1 knockout (KO) HeLa (F) and HCT116 (G) cells presented in . These cells were treated with WP1130 (10 μM) for the indicated periods before harvesting. (H and I) Treatment with the USP9X inhibitor WP1130 abrogates the anti-apoptotic effect of acetylation-mimetic MCL1 K40Q. These cells were treated with the indicated concentrations of WP1130 for 24 h and then subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates. Data in (A), (B), (F), and (G) are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Derivative Assay, Transfection, Construct, Ubiquitin Proteomics, Stable Transfection, Expressing, shRNA, CRISPR, Knock-Out

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) SIRT1, SIRT2, and SIRT3 efficiently deacetylate MCL1 in vitro . Shown is IB analysis of MCL1 acetylation after the in vitro deacetylation reaction and WCLs derived from HeLa cells transfected with the indicated Myc-MCL1 and FLAG-SIRT constructs for MCL1 and SIRT protein purification by FLAG and Myc immunoprecipitates . (B) MCL1 interacts with SIRT3 at the endogenous level. Shown is IB analysis of WCLs (input) and anti-MCL1 immunoprecipitates derived from 293T cells. (C) SIRT3 depletion results in the accumulation of MCL1 protein abundance. Shown is IB analysis of WCLs derived from HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2 , or SIRT3 . (D) SIRT3 depletion extends MCL1 protein half-life. HeLa cells stably expressing the lentiviral shRNA specific for GFP or SIRT3 presented in (C) were treated with 100 μg/mL CHX for the indicated period before harvesting. (E) SIRT3 depletion results in impairment of MCL1 poly-ubiquitination. HeLa cells stably expressing the lentiviral shRNA specific for GFP, SIRT1, SIRT2 , or SIRT3 were transfected with Myc-MCL1 and His-Ub constructs. 36 h after transfection, cells were treated with MG132 (10 μM) overnight before harvesting. (F) SIRT3 depletion accumulates endogenous Ac-K40-MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from HeLa cells stably expressing the indicated lentiviral shRNA specific for GFP or SIRT3 . (G) Ectopic SIRT3 expression increases ubiquitination of WT MCL1 but not K40R. 293T cells were transfected with Myc-MCL1, FLAG-SIRT3, and His-Ub constructs as indicated. 36 h after transfection, the cells were treated with MG132 (20 μM) for 5 h before harvesting. Left: His-Ub-conjugated proteins were captured with Ni-NTA agarose beads. Right: a schematic model of SIRT3-mediated MCL1 ubiquitination through K40 deacetylation followed by USP9X dissociation. (H and I) SIRT3 depletion confers resistance to doxorubicin-induced apoptosis through MCL1 stabilization. CRISPR-Cas9-mediated MCL1 KO and its parental HeLa cells were infected with the lentiviral shRNA specific for GFP or SIRT3 . These cells were treated with the indicated concentrations of doxorubicin for 24 h and then subjected to IB analysis (H) and a cell viability assay (I). Data are presented as mean ± SD; n = 3 biological replicates; **p < 0.01, ***p < 0.001. Data in (A)–(H) are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: In Vitro, Derivative Assay, Transfection, Construct, Protein Purification, Quantitative Proteomics, Stable Transfection, Expressing, shRNA, Ubiquitin Proteomics, CRISPR, Infection, Viability Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: (A) MCL1 acetylation levels correlate with p300 expression in breast and prostate cancer cell lines. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from a panel of breast and prostate cancer cell lines. (B) p300 depletion impairs MCL1 protein abundance in breast cancer cells with high p300 and acetylated MCL1 levels. Shown is IB analysis of WCLs derived from breast cancer cell lines stably expressing the lentiviral shRNA specific for GFP or p300 . (C) Treatment with the p300/CBP inhibitor A-485 results in decreased MCL1 protein levels in breast and prostate cancer cells with high p300 and acetylated MCL1 levels. Shown is IB analysis of WCLs derived from breast and prostate cancer cell lines treated with A-485 (3 μM) for 48 h before harvesting. (D) Representative images of MCL1 and p300 expression in breast tumor cells as assessed by immunohistochemistry (IHC). MCL1 and p300 levels were classified as low, moderate, or high, based on the intensities of the IHC staining, and a Spearman correlation test was conducted. Scale bar, 50 μm. See also .) (E and F) Treatment with the p300/CBP inhibitor A-485 reduces MCL1 acetylation and promotes dissociation of USP9X from MCL1. Shown is IB analysis of WCLs and anti-MCL1 immunoprecipitates derived from SKBR3 (E) and BT-20 (F) treated overnight with the indicated concentrations of A-485 before harvesting. (G and H) The USP9X inhibitor WP1130 effectively induces activation of the apoptotic pathway in cells with higher levels of acetylated MCL1. Shown is IB analysis of WCLs derived from the indicated breast cancer (G) and prostate cancer (H) cell lines. These cells were treated with WP1130 (5 μM) for the indicated periods before harvesting. (I and J) High acetylated MCL1 levels correlate with increased sensitivity to WP1130 in breast and prostate cancer cell lines. Breast cancer (I) and prostate cancer (J) cell lines were treated with the indicated concentrations of WP1130 for 24 h and subjected to cell viability assays. Data are presented as mean ± SD; n = 3 biological replicates; ***p < 0.001. Data in (A)–(C) and (E)–(H) are representative of at least two independent experiments. See also .

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Expressing, Derivative Assay, Quantitative Proteomics, Stable Transfection, shRNA, Immunohistochemistry, Activation Assay

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: MCL1 is an unstable protein that is targeted for ubiquitination. p300-directed MCL1 acetylation at K40 leads to enhanced interaction with USP9X, facilitating MCL1 deubiquitination and stabilization. SIRT3 is the potential deacetylase counteracting the acetylation-dependent MCL1 stabilization. Phosphorylation also plays important roles in regulating the interaction with USP9X and certain E3s. Elevated MCL1 acetylation status may correlate with sensitivity to USP9X and p300 inhibitors.

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Ubiquitin Proteomics, Histone Deacetylase Assay, Phospho-proteomics

Journal: Cell reports

Article Title: Interplay between protein acetylation and ubiquitination controls MCL1 protein stability

doi: 10.1016/j.celrep.2021.109988

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, a section was incubated with mouse monoclonal anti-p300 antibody diluted 1:150 (abcam54984, Cambridge, MA, USA) or rabbit polyclonal anti-MCL1 antibody diluted 1:100 (Proteintech) overnight at 4°C.

Techniques: Purification, Virus, Recombinant, Mutagenesis, Extraction, Cell Fractionation, Mass Spectrometry, Western Blot, CRISPR, Software

Journal: Cancer cell

Article Title: Cancer Cells Co-opt the Neuronal Redox-Sensing Channel TRPA1 to Promote Oxidative-Stress Tolerance

doi: 10.1016/j.ccell.2018.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal antibody anti-MCL-1 , Cell Signaling Technology , Cat# 5453S; RRID: AB_10694494.

Techniques: Recombinant, Membrane, Protein Extraction, Calcium Assay, Staining, Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, shRNA, Control, Software

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet: ( A, B ) Intracellular flow cytometry analysis of Bcl-xL ( B ) and Mcl-1 ( C ) expression in Fr. C cells (B220 + CD19 + CD43 + BP-1 + ) from wild-type (WT) and Mb1-cre ABCB7 conditional knockout (cKO) mice. Quantification of MdFI is shown on the right of each plot. Isotype controls are shown in gray. Offset histograms are representative of at least three independent experiments (total of 4–10 mice/group). ( C ) Flow cytometry analysis of Annexin V binding and fixable viability dye (FVD) labeling of Fr. B-C’ cells from WT and Mb1-cre ABCB7 cKO mice. Quantification of the proportion of each fraction that are dead (Annexin V + FVD + ) is shown on the right. Contour plots are representative of four independent experiments (total of 8–10 mice/group). ( D ) Quantification of the proportion of each fraction that are apoptotic (Annexin V + FVD - ) from ( C ). ( E ) Quantification of the proportion of each fraction that are apoptotic (Annexin V + FVD - ) after 16 hr in culture. Data represent three independent experiments (total of six mice/group). ( A–E ) Error bars represent SEM, and p-values are indicated above the data. Statistics were obtained by using an unpaired Student’s t -test.

Article Snippet: Antibody , Anti-Mcl-1 PE (Rabbit monoclonal, D2W9E) , Cell Signaling Technology , Cat#:65617S; RRID: AB_2799688 , FC (1:50).

Techniques: Flow Cytometry, Expressing, Knock-Out, Binding Assay, Labeling

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Mcl-1 PE (Rabbit monoclonal, D2W9E) , Cell Signaling Technology , Cat#:65617S; RRID: AB_2799688 , FC (1:50).

Techniques: Transgenic Assay, Selection, Concentration Assay, Sequencing, Recombinant, Flow Cytometry, EdU Assay, DNA Purification, Isolation, Colony-forming Unit Assay, Cell Cycle Assay, Software

Journal: Nature Communications

Article Title: Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors

doi: 10.1038/s41467-019-12409-w

Figure Lengend Snippet: Low BCL-X L expression in melanoma biases the pro-survival pool towards MCL1. a – c RNA-sequencing mRNA expression data obtained from the Cancer Cell Line Encyclopaedia for MCL1 ( a ) BCL-X L ( BCL2L1 ) ( b ) and MCL1 : BCL-X L ratio ( c ) in colorectal cancer (CRC), melanoma, non-small cell lung cancer (NSCLC) and pancreatic tumour cells. Expression values are reads per kilobase million (RPKM) and are presented on a log 2 scale. Black lines indicate median values. d , e Seven CRC (left) and seven melanoma (right) cell lines were cultured for 24 h before lysis and western blotting with the indicated antibodies. Quantification ( e ) of MCL1 and BCL-X L levels relative to A375 cells was performed by quantitative western blotting with fluorescently labelled secondary antibodies. f , g MCL1 ( f ) and BCL-X L ( g ) expression were quantified absolutely in CRC, melanoma, NSCLC and pancreatic tumour cell lines using recombinant protein standards and quantitative western blotting with fluorescently labelled secondary antibodies. Each point represents mean expression in a cell line from three independent experiments and is expressed in fmol per µg of total cellular protein (fmol µg −1 ). Black lines indicate median values. h Ratio of MCL1 to BCL-X L protein calculated from f and g , with black lines indicating median values. i Seven CRC (left) and seven melanoma (right) patient-derived xenograft (PDX) tumours were homogenised in lysis buffer and western blotted with the indicated antibodies. j , k Absolute quantification of MCL1 ( j ) and BCL-X L ( k ) expression in CRC and melanoma PDX tumour samples using recombinant protein standards and quantitative western blotting with fluorescently labelled secondary antibodies. Each point represents mean expression in a cell line from at least five technical replicates and is expressed in fmol per µg of total cellular protein (fmol µg −1 ). Black lines indicate median values. l Ratio of MCL1 to BCL-X L protein calculated from ( j ) and ( k ), with black lines indicating median values. Results ( e – h , j – l ) are the mean of three or more independent experiments, and error bars indicate SD. P ≤ 0.0001 (****), P ≤ 0.001 (***), P ≤ 0.01 (**), P ≤ 0.05 (*), ns (not significant) as determined by Kruskal–Wallis and Dunn’s multiple comparisons tests ( a – c , f – h ) or two-tailed Mann–Whitney test ( j – l )

Article Snippet: Recombinant protein standards for MCL1 (Cloud-Clone Corp, Texas, USA) and BCL-X L (R&D Systems, Abingdon, Oxfordshire, UK) were diluted in RIPA with 1 × Laemmli sample buffer to a concentration range of 2.5–100 nM.

Techniques: Expressing, RNA Sequencing Assay, Cell Culture, Lysis, Western Blot, Recombinant, Derivative Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors

doi: 10.1038/s41467-019-12409-w

Figure Lengend Snippet: ERK1/2 pathway inhibitors combine with the MCL1 inhibitor AZD5991 to kill melanoma cells. a – c A375 ( a ), SK-MEL-28 ( b ) and WM266-4 ( c ) cells were treated with the indicated concentrations of trametinib or vemurafenib in combination with AZD5991 for 5 days. The number of viable cells was determined at the point of treatment (day 0) and at the end of the experiment using Sytox Green. One-hundred percent represents the number of viable cells in the control wells at day 5, 0% is equivalent to the number of viable cells on day 0 and −100% indicates that no viable cells remained on day 5. d , e Twelve melanoma and 12 colorectal cancer (CRC) cell lines were treated with trametinib (Tra) or selumetinib (Sel) and AZD5991 or AZD4320 in an 8 × 8 concentration matrix for 5 days. Viable cell number (determined as above) was used to calculate Loewe synergy scores using the Loewe additivity model. P ≤ 0.01 (**), P ≤ 0.05 (*), ns (not significant) as determined by unpaired or paired two-tailed t -test. f A375 cells were treated with increasing concentrations of vemurafenib (Vem) with or without 1 μM AZD5991 for 48 h. Cell cycle profile was determined by propidium iodide staining and flow cytometry. g , h A375 cells were treated with increasing concentrations of vemurafenib (Vem) ( g ) or selumetinib (Sel) ( h ) with or without 1 μM AZD5991 for 48 h. i The indicated tumour cell lines were treated with DMSO control (C), 1 μM vemurafenib (Vem), 1 μM selumetinib (Sel) and 0.3 μM SCH772984 (SCH) alone or in combination with 1 μM AZD5991, 1 μM AZD4320 and 20 μM Q-VD-OPh (QVD) for 48 h. j – o A375 ( j , k ), COLO205 ( l , m ) or SW620 ( n , o ) cells were treated with increasing concentrations of AZD5991 ( j , l , n ) or AZD4320 ( k , m , o ) with or without 1 μM selumetinib (Sel) for 48 h. p , q A375 ( p ) and SK-MEL-30 ( q ) cells were treated with increasing concentrations of A-1155463 with or without 1 μM selumetinib (Sel) and 3 μM venetoclax for 48 h. g – q Apoptosis was assessed by Annexin V positivity using flow cytometry. Results ( a – q ) are the mean of at least three independent experiments and error bars show SD

Article Snippet: Recombinant protein standards for MCL1 (Cloud-Clone Corp, Texas, USA) and BCL-X L (R&D Systems, Abingdon, Oxfordshire, UK) were diluted in RIPA with 1 × Laemmli sample buffer to a concentration range of 2.5–100 nM.

Techniques: Concentration Assay, Two Tailed Test, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors

doi: 10.1038/s41467-019-12409-w

Figure Lengend Snippet: Apoptosis induced by combined ERK1/2 pathway and MCL1 inhibition requires BIM and BMF. a A375 cells were treated with the indicated concentrations of vemurafenib (Vem) with or without 1 μM AZD5991 for 24 h. b SK-MEL-30 cells were treated with the indicated concentrations of selumetinib (Sel) with or without 1 μM AZD5991 for 24 h. c A375 cells were treated with the indicated concentrations of AZD5991 with or without 1 μM vemurafenib (Vem) for 24 h. d SK-MEL-30 cells were treated with the indicated concentrations of AZD5991 with or without 1 μM selumetinib (Sel) for 24 h. a – d Whole-cell lysates were western blotted with the indicated antibodies. e , f A375 cells and three independent clonally derived WT, BIM KO, BMF KO and BIM BMF DKO A375 CRISPR clones were treated with 1 μM selumetinib (Sel) as indicated for 24 h and BIM and BMF expression assessed by western blotting ( e ) or treated with DMSO-only control (C), 1 μM selumetinib (Sel), 1 μM AZD5991 or 1 μM selumetinib plus 1 μM AZD5991 (Sel + AZD5991) as indicated for 48 h and apoptosis assessed by Annexin V staining and flow cytometry ( f ). Results ( f ) show mean ± SD of three independent experiments. P ≤ 0.0001 (****), P ≤ 0.01 (**) and ns (not significant) as determined by two-way ANOVA and Tukey’s multiple comparisons test. g , h SK-MEL-30 cells and three independent clonally derived WT and BIM BMF DKO SK-MEL-30 CRISPR clones were treated with 1 μM selumetinib (Sel) as indicated for 24 h and BIM and BMF expression assessed by western blotting ( g ) or treated with DMSO-only control (C), 1 μM selumetinb (Sel), 1 μM AZD5991 or 1 μM selumetinib plus 1 μM AZD5991 (Sel + AZD5991) as indicated for 48 h and apoptosis assessed by Annexin V staining and flow cytometry ( h ). Results ( h ) show mean ± SD of three independent experiments. P ≤ 0.0001 (****) as determined by two-way ANOVA and Tukey’s multiple comparisons test

Article Snippet: Recombinant protein standards for MCL1 (Cloud-Clone Corp, Texas, USA) and BCL-X L (R&D Systems, Abingdon, Oxfordshire, UK) were diluted in RIPA with 1 × Laemmli sample buffer to a concentration range of 2.5–100 nM.

Techniques: Inhibition, Western Blot, Derivative Assay, CRISPR, Clone Assay, Expressing, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors

doi: 10.1038/s41467-019-12409-w

Figure Lengend Snippet: Combined BRAFi and MCL1i redistributes BIM and BMF to other pro-survival proteins. a A375 cells were treated with DMSO vehicle-only (C and 5991) or 2 μM vemurafenib (Vem and Vem + 5991) for 24 h followed by DMSO-only (C and Vem) or 1 μM AZD5991 (5991 and Vem + 5991) for a further 4 h. Lysates (input) were subjected to immunoprecipitation with antibodies to BCL-X L or MCL1. Input lysates, immunoprecipitates (IP) and supernatant unbound fractions (UNB) were then western blotted using the indicated antibodies. b A375 cells were treated with DMSO vehicle-only (C and 1155) or 2 μM vemurafenib (Vem and Vem + 1155) for 24 h followed by DMSO-only (C and Vem) or 0.1 μM A-1155463 (1155 and Vem + 1155) for a further 4 h. Lysates (input) were subjected to immunoprecipitation with antibodies to BCL-X L or MCL1. Input lysates, immunoprecipitates (IP) and supernatant unbound fractions (UNB) were then western blotted using the indicated antibodies

Article Snippet: Recombinant protein standards for MCL1 (Cloud-Clone Corp, Texas, USA) and BCL-X L (R&D Systems, Abingdon, Oxfordshire, UK) were diluted in RIPA with 1 × Laemmli sample buffer to a concentration range of 2.5–100 nM.

Techniques: Immunoprecipitation, Western Blot

Journal: Cancer cell

Article Title: Treatment-induced tumor dormancy through YAP-mediated transcriptional reprogramming of the apoptotic pathway

doi: 10.1016/j.ccell.2019.12.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal (D5V5L) anti-MCL1 , Cell Signaling , Cat#39224; RRID: AB_2799149.

Techniques: Recombinant, In Vivo, In Vitro, Synthesized, Control, Reverse Transcription, Gene Expression, Staining, Cell Viability Assay, TaqMan Assay, Plasmid Preparation, Software

Journal: Cell Reports Medicine

Article Title: Venetoclax acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against leukemia

doi: 10.1016/j.xcrm.2024.101580

Figure Lengend Snippet:

Article Snippet: Anti-human MCL-1-Alexa Flour 647 (D2W9E) , Cell Signaling Technology , Cat# 78471; RRID: AB_331284.

Techniques: Recombinant, Staining, Blocking Assay, CCK-8 Assay, Western Blot, Cell Isolation, Software

Journal: Cell Reports Medicine

Article Title: Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity

doi: 10.1016/j.xcrm.2020.100188

Figure Lengend Snippet:

Article Snippet: Mcl-1 Rabbit mAb D2W9E , Cell Signaling , Cat # 94296; RRID: AB_2722740.

Techniques: Recombinant, Cell Viability Assay, Caspase-Glo Assay, Isolation, SYBR Green Assay, Software, Empire Assay