Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: The expression of Beclin-1 is reduced in a fraction of human colorectal cancers. Beclin-1 expression was analyzed by immunohistochemistry on the tissue microarray carrying patient-derived colorectal cancer tissue. A, representative images of colorectal cancers expressing high (left) and low (right) levels of Beclin-1. B, quantification of the Beclin-1 immunostaining intensity in human colorectal cancer samples. Cancer sample numbers shown on the figure are the same as on the original array. The intensity of staining was scored from 0 to 3 (0 for negative, 1 for weak, 2 for moderate, and 3 for strong staining). The staining index (H-score) was generated according to the formula H-score = 0xf0 + 1xf1 + 2xf2 + 3xf3, where 0, 1, 2, and 3 are staining intensity and f0 through f3 are the fractions of the cells showing staining intensity from 0 to 3, respectively. The data represent the average of two independent experiments (performed on two different serial sections) plus the S.D. Student's one-sample t test was used to measure the variance of Beclin-1 staining indexes among the samples derived from different patients and identify those tumors for which these indexes varied significantly from the mean value (204.59) observed for the whole population of tumor samples. The lowest index for samples that did not differ from the mean value within the 95% confidence interval was 179.39. Values that varied significantly (p < 0.0001) from the mean are indicated with an asterisk. These values ranged from 0 to 150. C, indicated human colon carcinoma-derived cell lines carrying wild type (wt) or oncogenic (onc) K-ras were assayed for Beclin-1 expression by Western blot. α-Tubulin was used as a loading control.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Expressing, Immunohistochemistry, Microarray, Derivative Assay, Immunostaining, Staining, Generated, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: The reversal of ras-induced down-regulation of Beclin-1 blocks proliferation of detached intestinal epithelial cells. A and B, indicated cell lines were plated attached to the ECM or in soft agar in the absence (−) or in the presence (+) of 8 ng/ml tetracycline, cell colonies were allowed to form for 7–10 days and counted. The results are expressed as a percentage of cells initially plated. The data represent the average of the duplicates plus the S.D. This experiment was repeated twice with similar results. C and D, indicated cell lines were plated attached to or detached from the ECM for 96 h in the absence (−) or in the presence (+) of 8 ng/ml tetracycline, and cells were counted. The results are presented as the number of cells observed at the indicated time less the number of cells initially plated. The data represent the average of the triplicates plus the S.E. This experiment was repeated twice with similar results. E and F, indicated cell lines were plated attached to or detached from the ECM for 72 h in the absence (−) or in the presence (+) of 8 ng/ml tetracycline, and their cell cycle profile was analyzed by flow cytometry. The data in E represent the average of the duplicates plus the S.D. This experiment was repeated twice with similar results. The data in F represent the average of two independent experiments plus the S.D. *, indicates that the p value was <0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Oncogenic ras triggers down-regulation of Beclin-1 in intestinal epithelial cells. A, attached (att) and detached (det) IEC-18 cells and two independently derived H-ras-transformed clones of these cells ras-3 and ras-7 were assayed for the expression of Beclin-1 by Western blot. B, attached MT-ras cells were cultured in the absence (−) and in the presence (+) of 100 μm Zn2+ and 2 μm Cd2+ for 24 h (left) and 48 h (right) and assayed for the expression of Beclin-1 by Western blot. C, IEC-18 cells were processed as in B. D, attached human colorectal carcinoma cells HCT-116 and their K-ras knock-out derivatives Hkh-2 and Hke-3 were assayed for the expression of Beclin-1 by Western blot. The membranes in A and D were re-probed with a CDK-4 antibody, and the membranes in B and C were re-probed with an anti-α-tubulin as loading controls.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Derivative Assay, Transformation Assay, Clone Assay, Expressing, Western Blot, Cell Culture, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: The reversal of ras-induced down-regulation of Beclin-1 promotes the formation of autophagic vacuoles following detachment of intestinal epithelial cells. A, indicated cell lines were assayed for Beclin-1 expression by using a Beclin-1-specific antibody by Western blot in the absence (−) or in the presence (+) of 8 ng/ml tetracycline. The positions of endogenous (endog.) Beclin-1 and exogenous (exog.) FLAG-tagged Beclin-1 on the gel are indicated. CDK-4 was used as a loading control. B, cells were cultured attached to detached from the ECM in the absence (−) or in the presence (+) of 8 ng/ml tetracycline for 48 h and analyzed for the presence of autophagic vacuoles as in Fig. 3B. Percentage of cells with punctuate GFP-LC3 distribution observed in attached cells was subtracted from that observed in detached cells as background. The data represent the average of the duplicates plus the S.D. This experiment was repeated twice with similar results. *, p value < 0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Expressing, Western Blot, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Enforced down-regulation of Beclin-1 increases the ability of detached non-malignant intestinal epithelial cells to enter the S phase of the cell cycle. IEC-18 (A), ras-3 (B), or IEC-Bcl-X cells (F) were cultured attached to or detached from the ECM for 24 h and analyzed for the cell cycle profile by flow cytometry. C, IEC-18 cells were transfected with a control non-targeting RNA (cont RNA) or Beclin-1-specific siRNA-1 (Beclin siRNA-1) or Beclin-1-specific siRNA-2 (Beclin siRNA-2) and analyzed for Beclin-1 expression by Western blot. CDK-4 was used as a loading control. D, cells processed as in C were analyzed as in A. E, IEC-18 or IEC-BclX cells transfected with the indicated RNAs as in C, were then plated in monolayer either immediately or after being cultured in suspension for 26 h and allowed to form colonies, which were counted 7–10 days later. Results are expressed as a percentage of the number of colonies obtained after culturing cells in monolayer immediately after transfection. The data in D represent the average of two independent experiments plus the S.D. The data in A, B, E, and F represent the average of the duplicates plus the S.D. All experiments were repeated twice with similar results. *, p value <0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Cell Culture, Flow Cytometry, Transfection, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Oncogenic ras reduces the stability of Beclin-1 in intestinal epithelial cells. A, attached IEC-18 and ras-3 cells were assayed for Beclin-1 expression by Northern blot. 18 S and 28 S ribosomal RNAs were used as loading controls. B, IEC-18 (top) and ras-3 (bottom) cells were cultured in the presence of 35S-labeled methionine and cysteine, which were then diluted with respective unlabeled amino acids, cells were collected at the indicated times, Beclin-1 was immunoprecipitated from the cells, and Beclin-1 levels were analyzed by electrophoresis in polyacrylamide gel followed by autoradiography. The contrast of the image corresponding to ras-3 cells was increased to illustrate the fact that the rate of reduction of Beclin-1 levels in ras-3 cells is higher than that in IEC-18 cells. C, cells were processed as in A, and Beclin-1 levels were quantified by phosphorimaging and densitometry. Beclin-1 level in each cell line at each time point is expressed as a percentage of that observed in the same cell line at 0-h chase. The data represent the average of the duplicates plus the S.D. This experiment was repeated twice with similar results. *, p value <0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Expressing, Northern Blot, Cell Culture, Labeling, Immunoprecipitation, Electrophoresis, Autoradiography

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Oncogenic ras down-regulates Beclin-1 in intestinal epithelial cells in a calpain-dependent manner. A, ras-3 cells were cultured attached to the ECM and treated with 10 μm ALLM (left) for 0 h (−) or 24 h (+) or 60 μm PD150606 (right) for 0 h (−) or 4 h (+) and analyzed for Beclin-1 expression by Western blot. CDK-4 was used as a loading control. Treatment with vehicle (DMSO) did not increase Beclin-1 expression in ras-3 cells under the conditions indicated above (not shown). B, IEC-18 cells were processed as in A. C, IEC-18 and ras-3 cells were cultured attached to the ECM for 24 h and assayed for calpain activity. Calpain activity observed in ras-3 cells was arbitrarily designated as 1.0. D, tet-ras-Beclin cells were treated with 800 ng/ml tetracycline for 24 h (to generate Beclin-1 in quantities sufficient for further analysis), Beclin-1 was immunoprecipitated from these cells, the immunoprecipitated material was not treated (−) or treated (+) with calpain-1 and analyzed for Beclin-1 expression by Western blot. E, ras-3 cells were analyzed for the ability to form colonies in monolayer culture (attached) or in soft agar (soft agar) as in Fig. 5A in the absence (−) and in the presence (+) of 60 μm PD150606. The data in C and E represent the average of the duplicates plus the S.D. These experiments were repeated twice with similar results. *, p value <0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Cell Culture, Expressing, Western Blot, Activity Assay, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Oncogenic ras triggers calpain activity and down-regulates Beclin-1 in intestinal epithelial cells in a RhoA-dependent manner. A, IEC-18 and ras-3 cells were cultured attached to the ECM for 24h, respective cell lysates were incubated with glutathione-agarose linked to the Rho binding domain of Rhotekin (Rho binding partner), and RhoA captured in this manner was detected by Western blot. B and D, ras-3 cells were transfected with a control non-targeting RNA (cont RNA) or RhoA-specific siRNA-1 (RhoA siRNA-1) or RhoA-specific siRNA-2 (RhoA siRNA-2) and analyzed for RhoA (B) or Belin-1 (D) expression by Western blot. β-Actin was used as a loading control. C, ras-3 cells were processed as in B and analyzed for calpain activity as in Fig. 8B. Calpain activity observed in ras-3 cells that were transfected with a control RNA was arbitrarily designated as 1.0. The data represent the average of the duplicates plus the S.D. This experiment was repeated twice with similar results. *, p value <0.05.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Activity Assay, Cell Culture, Incubation, Binding Assay, Western Blot, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Oncogenic ras -induced Down-regulation of Autophagy Mediator Beclin-1 Is Required for Malignant Transformation of Intestinal Epithelial Cells *

doi: 10.1074/jbc.M109.046789

Figure Lengend Snippet: Schematic representation of the mechanism by which oncogenic Ras promotes proliferation of malignant intestinal epithelial cells in the absence of adhesion to the ECM. Oncogenic Ras activates RhoA, which in turn increases cellular calpain activity. The latter event leads to the degradation of Beclin-1 and subsequent increase of the ability of oncogenic Ras-carrying malignant cells to proliferate without being attached to the ECM.

Article Snippet: The binding of the anti-Beclin-1 antibody to Beclin-1 was visualized using 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Inc.).

Techniques: Activity Assay

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Schematic for the surface IP of BCL2-family proteins. Anti-apoptotic proteins in cell extracts were immobilized onto the surface of the reaction chamber. b , Schematic of immunoassay selectively detecting protein complexes (left) and the total level of surface bait protein (right). The fluorescence signals from detection antibodies were imaged under total internal reflection (TIR) excitation. c , Comparison of the formation of model BCL2-BIM BH3 complex in an intracellular environment by BCL2-mCherry/BIM BH3 -eGFP co-expression (right) and in vitro mixing of two individual extracts with each expressing BCL2-mCherry and BIM BH3 -eGFP (left) ( n = 10 independent images). d , Changes in active BAX level measured for staurosporine-treated HL60 cells after lysis and IP with different detergent types ( n = 5 independent images). e , Single-molecule count of surface-immobilized BCL2-eGFP from the crude extract with indicated IP antibodies ( n = 10 independent images). f , Comparison of signal-to-noise ratio between single-molecule counting analysis and integration of total fluorescence signals. g , Counts of endogenous BCL2 protein complexes from the 30,000 HL60 cells by using immunoassay ( n = 3 biological replicates) (two-sided two-sample t -test, *** P = 1.5 × 10 −5 , 1.1 × 10 −5 ). h , Counts of endogenous BCL2-BIM complexes and interchip c.v.s from the fixed numbers of HL60 cells ( n = 3 biological replicates). i , Interchip c.v.s obtained from independent interchip measurement for all immunoassays and cell numbers ( n = 3 biological replicates). Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Fluorescence, Comparison, Expressing, In Vitro, Lysis, Single Molecule Counting

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a-c , Selection of monoclonal antibodies for surface immunoprecipitation (IP). Surface IP of eGFP-labeled target anti-apoptotic proteins from transfected HEK293T crude cell extracts. Crude cell extracts with 1 nM of eGFP-labeled anti-apoptotic protein were used to IP. ( a ) BCL2 IP antibody, ( b ) BCLxL IP antibody, ( c ) MCL1 IP antibody ( n = 10 independent images). d-f , Selection of monoclonal antibodies for total level immunoassay of surface-immobilized target anti-apoptotic proteins. ( d ) BCL2 detection antibody, ( e ) BCLxL detection antibody, ( f ) MCL1 detection antibody ( n = 10 independent images). g , Relative model BCL2-BIM BH3 complex from BCL2-mCherry/BIM BH3 -eGFP co-transfected HEK293T cells after the lysis with different detergent types ( n = 10 independent images). h , Detection of surface-immobilized BAK fragments by using the monoclonal antibody AT38E2. Crude cell extracts with 1 nM of eGFP-labeled BAK fragments were used to IP ( n = 10 independent images). The data were normalized to the BAK level of 24-69 (α1) fragments. i , Activation of BAK from HL60 and Ramos cells by heat or triton X-100 treatment to crude cell extracts. The data were normalized to the active BAK level of non-treated HL60. j , Schematics for the preparation of HL60 samples with different preservation conditions. k,l , Comparison of the PPI profiles across the sample states using SMPC platform. ( k ) BCL2 total level, ( l ) BCL2-BAX complex ( n = 10 independent images). Error bars represent means±s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Selection, Immunoprecipitation, Labeling, Transfection, Lysis, Activation Assay, Preserving, Comparison

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Setting the cutoff to eliminate background signals for post image analysis. We used 8 to 10 control images captured with a 100 ms time resolution from empty reaction chambers to establish a background limit. The background limit (cutoff=1,015 A.U.) was determined from the signal distribution recorded in each pixel of the control images, effectively eliminating 99% of background signals. b , Identification of diffraction-limited fluorescence spots. Three sequential images captured with a 300 ms time frame were averaged to create a single image upon subtracting the background. From the averaged image, the number of diffraction-limited fluorescence spots were counted (single-molecule counting). c , Identified single-molecule counts and integration of individual pixel signals of the imaging area (total fluorescence integration) from the analyzed TIRF images. The TIRF images were obtained by surface IP of BCLxL-eGFP expressed in HEK293T cell extracts (Scale bar: 10 μm) ( a-c ). d-g , Calibration of single-molecule count from the total fluorescence integration. ( d ) Single-molecule count dependent on the total fluorescence integration of surface-immobilized BCL2-mCherry obtained from the analyzed images (Scale bar: 10 μm). ( e ) Photobleaching kinetics of surface-immobilized BCL2-mCherry signals depending on the bleaching time. ( f ) The linear correlation between single-molecule count and total fluorescence integration of surface-immobilized BCL2-mCherry after photobleaching. ( g ) Calibration of single-molecule count from total fluorescence integration by extrapolating. The linear correlation was obtained in ( f ). The TIRF images were obtained by surface IP of BCL2-mCherry expressed in HEK293T cell extracts ( d-g ).

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Control, Fluorescence, Single Molecule Counting, Imaging

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Counts of BCL2-related immunoassays (BCL2-BIM complex, BCL2-BAX complex, BCL2 total level) from the fixed numbers of HL60 cells ( n = 3). b , Counts of BCLxL-related immunoassays (BCLxL-BIM complex, BCLxL-BAX complex, BCLxL-BAD complex, BCLxL-BAK complex, BCLxL total level) from the fixed numbers of PC9 cells ( n = 3). c , Counts of MCL1-related immunoassays (MCL1-BIM complex, MCL1-BAK complex, MCL1 total level) from the fixed numbers of PC9 cells ( n = 3). The single-molecule counts were rescaled to account for the labeling efficiencies of the immunoassays calculated in Extended Data Fig. as well as the specific incubation conditions for direct comparison. All data were measured from independent inter-chip experiments. CVs obtained from independent inter-chip measurement for all the immunoassays and cell numbers ( n = 3). Individual data points shown for independent biological replicates.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Labeling, Incubation, Comparison

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a – d , Changes in endogenous BCL2-family PPI profiles of HL60 cells through apoptosis progression by 2 μM of staurosporine. Active BAX/BAK level ( a ), total levels of anti-apoptotic proteins ( b ), BIM complexes ( c ), BAX complexes ( n = 10 independent images) ( d ). e , Schematic of the PBA to measure the unoccupied populations of surface-immobilized anti-apoptotic proteins. f , Changes in BIM BH3 PBAs for anti-apoptotic proteins from HL60 cells through apoptosis progression by staurosporine ( n = 10 independent images). g , Schematic of the comparison of the BCL2 protein complex compositions in different AML cell lines (left). The density of surface-immobilized BCL2 was constantly maintained by optimizing the total protein concentration of crude cell extracts in all AML cell lines (right) ( n = 10 independent images). h – j , Compositions of the BCL2 PPI profiles in AML cell lines. BCL2 protein complexes ( h ), BCL2-BIM BH3 PBA ( n = 10 independent images) ( i ), integrated BCL2 complex compositions ( j ). k , Changes in BCL2-BIM BH3 PBA and BCL2-BAD PBA with increasing amounts of BAD probe ( n = 10 independent images). BIM BH3 probe was presented at 10 nM. The single-molecule counts were rescaled to account for the labelling efficiencies of the immunoassays calculated in Extended Data Fig. . Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Comparison, Protein Concentration

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Comparing relative efficiencies of IP antibodies for anti-apoptotic proteins. 1 nM of eGFP-labeled anti-apoptotic proteins were immobilized on the surface by the matched IP antibodies respectively, and the eGFP signals were compared ( n = 10 independent images). b , Comparing relative efficiencies of immunolabeling antibodies for anti-apoptotic proteins. The surface immobilized anti-apoptotic protein in ( a ) were immunolabeled by the matched labeling antibodies respectively, and the relative efficiencies for total level assays were compared ( n = 10 independent images). c , Comparing relative immunolabeling efficiencies of detection antibodies for BCL2 family proteins. 1 nM of eGFP-labeled BCL2 family proteins were immobilized on the surface by the anti-eGFP antibody and detected with matched detection antibodies for immunolabeling. The relative immunolabeling efficiencies of the detecting antibodies were calculated based on the ratio of labeled proteins to the total number of proteins immobilized on the surface ( n = 10 independent images). The data were normalized to the efficiencies of BCL2 IP assay, BCL2 total level assay, and BCL2 immunolabeling antibody, respectively ( a-c ). Error bars represent means±s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Labeling, Immunolabeling

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a-d , PBA binding curves for each PPI probes (eGFP-labeled BIM BH3 , BIM EL , BAD, NOXA) to anti-apoptotic proteins (mCherry-labeled BCL2, BCLxL, MCL1) to calculate dissociation constant ( K d ). ( a ) BIM BH3 -PBA, ( b ) BIM EL -PBA, ( c ) BAD-PBA, ( d ) NOXA-PBA. e , Comparison of the binding affinities between each binding pairs. f , Correlations between the calculated K d values and the occupancy values at a fixed PPI probe concentration. g , Dissociation of BCL2-BIM complex after in vitro competition by BAD-eGFP PPI probes ( n = 10 independent images). Protein complexes were surface-immobilized by anti-BCL2 IP antibody and detected with anti-BIM detection antibody after the competition. h , Schematic of in vitro PBA competition between BIM BH3 -eGFP PPI probe and competitors (PPI probe or BH3 mimetics) on surface-immobilized BCL2 proteins. i , Remained BCL2-BIM BH3 PBA after in vitro binding competition with BH3 mimetics (ABT-199, AZD-5991, WEHI-539). BIM BH3 PPI probe was presented in 10 nM. j,k , BCL2 PBA counts with different PPI probes from four AML cell lines. ( j ) BCL2-BIM EL PBA, ( k ) BCL2-BAD PBA ( n = 10 independent images). l , Schematic for selective binding of PPI probes for unoccupied BCL2 proteins. Error bars represent means±s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Binding Assay, Labeling, Comparison, Concentration Assay, In Vitro

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a – e , Changes in BCL2-family PPI profiles in HL60 cells after ABT-199 treatment (100 nM, 24 h). Total levels of anti-apoptotic proteins ( a ), active BAX/BAK level ( b ), total levels of BAX/BAK ( c ), BCL2 complexes ( d ), BCL2-BIM BH3 PBA ( n = 10 independent images) ( e ). f – h , Changes in BCL2-family PPI profile in HL60 cells after higher concentration of ABT-199 treatment (300 nM, 4 h). BCL2 complexes ( f ), active BAX/BAK level ( g ), total levels of BAX/BAK ( n = 10 independent images) ( h ). i , Schematic illustration of the mode of action of ABT-199. j – n , Changes in BCL2-family PPI profiles in U937 cells after AZD-5991 treatment (100 nM, 24 h). Active BAX/BAK level ( j ), MCL1 complexes ( k ), MCL1-BIM BH3 PBA ( l ), BCLxL complexes ( n = 10 independent images) ( m ), comparison of relative changes in BAK complexes after AZD-5991 treatment ( n ). The relative changes were obtained from the data indicated in k and m normalized to BCLxL- or MCL1-total level. o , Schematic illustration of the mode of action of AZD-5991. The single-molecule counts were rescaled to account for the labelling efficiencies of the immunoassays calculated in Extended Data Fig. ( d , k , m ). Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Concentration Assay, Comparison

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , BIM BH3 PBA profiles of four different leukemia cell lines. The BIM BH3 PBA levels were rescaled to account for the relative PBAs of BIM BH3 probe for anti-apoptotic proteins determined in Extended Data Fig. . The data were normalized to the BCL2-BIM BH3 PBA level of HL60 cell. b,c , Viability of leukemia cell lines after treatment of BH3 mimetics for 24 hours. ( b ) ABT-199, ( c ) AZD-5991.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Schematic for generating linear regression correlation between ex vivo efficacy of ABT-199 and PPI profiles from primary AML samples. The collected AML cells were cultured and treated with 0–1 μM of ABT-199, and the AUCs of cell viability were obtained as ex vivo drug efficacy (top). Approximately 1.2 × 10 6 of primary AML cells on the same cohort underwent PPI profiling with the SMPC (bottom). b , The absolute Pearson correlations between ex vivo AUC of ABT-199 and PPI metrics for primary AML samples (one-sided F -test, * P < 0.05, ** P < 0.01, *** P < 0.001, NS, not significant; P values are provided as ) ( n = 32). c , d , Correlations between ex vivo AUC and single BCL2-related PPI metrics. BCL2-BAX CPX (coefficient: 0.157, P = 6.7 × 10 −8 ) ( c ), BCL2-BIM CPX (coefficient: 0.013, P = 0.01) (one-sided F -test) ( d ). e , Lasso coefficients of PPI profiles correlated with ex vivo AUC of ABT-199 for primary AML samples (67 models). f , Correlation between ex vivo AUC and the combination of multiple PPI metrics (BCL2-BIM BH3 PBA, BCL2-BAX CPX and BCLxL-BAK CPX) (one-sided F -test, P = 1.2 × 10 −10 ). g , ROC curve between the estimated score and the ex vivo efficacy (two-sided t -test, P = 2.9 × 10 −5 ). h , Clinical features and the ABT-199 administration history of BC-7064. i , Comparison of the PPI profiles and the estimated scores with PPI diagnostic results from the initial and the relapsed BC-7064 samples (R, responsive; NR, non-responsive) ( n = 10 independent images). Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Ex Vivo, Cell Culture, Comparison, Diagnostic Assay

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Comparison of BCL2 family PPI profiles from the healthy donor PBMC sample and HL60 cells with SMPC ( n = 10 independent images). The data were normalized to the HL60 cell. b , Correlation between ex vivo AUC for ABT-199 and combination of BCL2-related metrics (BCL2 total level, BCL2-BIM BH3 PBA, BCL2-BAD PBA, and BCL2-BAX CPX) (One-sided F -test, p -value = 1.3e-10). c , Linear correlations between two different BCL2 PBA metrics. d , Correlations between ex vivo AUC and IC 50 of primary AML samples for ABT-199 ( n = 32). e , Schematic of Lasso regression analysis for the selection of PPI metrics highly correlated with drug response. The training and test groups were randomly selected from the primary AML sample cohort. The Lasso regression models were generated using the training group and evaluated based on Pearson’s R as well as prediction outcomes for the test group. 67 models were selected from 10,000 different initial models. f , Identification of outliers in the ABT-199 drug efficacy prediction models. The residuals of each outlier in the model ( ex vivo AUC – estimated score) were indicated. g , Correlations between ex vivo AUC and IC 50 of primary AML samples for AZD-5991 ( n = 27). h , Correlation between ex vivo AUC for AZD-5991 and combination of MCL1-related metrics (MCL1 total level, MCL1-BIM BH3 PBA, MCL1-NOXA PBA, and MCL1-BAK CPX) (One-sided F -test, p -value = 2.5e-5).

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Comparison, Ex Vivo, Selection, Generated

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Schematic for BH3 profiling based on JC-1 staining, and measurement of BCL2 levels using flow cytometry or western blotting for primary AML samples. b,c , BH3 profiling on HL60 cells. ( b ) Relative fluorescence units (RFU) of 590 nm wavelength for HL60 cells through the treatment of BH3 peptides or BH3 mimetics, ( c ) Depolarization of HL60 cells through the BH3 profiling. Depolarization by 10 μM of BAD peptides was indicated. d,e , Correlations for ex vivo ABT-199 AUC with ( d ) depolarizations by BAD (10 μM) – HRK (10 μM) peptides, ( e ) combination of multiple PPI metrics (BCL2-BIM BH3 PBA, BCL2-BAX CPX, and BCLxL-BAK CPX). The outliers identified in model ( d ) were indicated ( n = 14) (One-sided F -test, p -value = 0.011, 8.1e-05). f,g , ROC curves for ex vivo ABT-199 responses with ( f ) depolarizations by BAD (10 μM) – HRK (10 μM) peptides, ( g ) Combination of multiple PPI metrics (Two-sided t -test, p -value = 0.07, 0.009). h , Correlations between BCL2 protein levels determined by SMPC and flow cytometry for primary AML samples (n = 14 ). i , Prediction models for ABT-199 ex vivo AUC with BCL2 protein levels determined by flow cytometry (One-sided F -test, p -value = 0.077). j , Correlations between BCL2 protein levels determined by SMPC and western blotting for primary AML samples ( n = 32). The raw blot image is provided as a Source Data. k , Prediction models for ABT-199 ex vivo AUC with BCL2 protein levels determined by western blotting (One-sided F -test, p -value = 0.0003).

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Staining, Flow Cytometry, Western Blot, Fluorescence, Ex Vivo

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a , Schematic of the study. Complete blood cell count analysis was carried out on a daily basis to count the number of AML blasts in the primary AML samples. b – d , Comparison of the PPI profiles from the BC-6524 and BC-7230 samples. Clinical features and the estimated scores with PPI diagnostic results for ABT-199 ( b ), comparison of BCL2-family PPI profiles ( n = 10 independent images) ( c ), changes in in vivo AML blast counts through the days after ABT-199 administration ( d ). e , Confusion matrix for the drug response prediction based on the estimated score and the in vivo drug responses ( n = 10). f , Comparison of the estimated scores of patients with AML between responsive ( n = 4) and non-responsive ( n = 6) patients for in vivo ABT-199 administration (two-sided Mann–Whitney test, * P = 0.014). For the boxplots, the centre line represents the median, the box limits are the upper and lower quartiles, and the whiskers represent 1.5× interquartile range. g , Clinical features and the ABT-199 administration history of BC-7107. h , Tracking the changes in BCL2-family PPI profiles after relapse of BC-7107-R ( n = 10 independent images). i , Tracking the changes in BIM BH3 PBA profiles and PPI diagnostic results after relapse of BC-7107-R. The data were normalized to the BCL2-BIM BH3 PBA level of HL60 cells. j , Changes in in vivo AML blast counts from BC-7107-R through the days after ABT-199 administration. The estimated scores were calculated from the model in Fig. ( b , g ). Data represent means ± s.d.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: Cell Counting, Comparison, Diagnostic Assay, In Vivo, MANN-WHITNEY

Journal: Nature Biomedical Engineering

Article Title: Profiling protein–protein interactions to predict the efficacy of B-cell-lymphoma-2-homology-3 mimetics for acute myeloid leukaemia

doi: 10.1038/s41551-024-01241-3

Figure Lengend Snippet: a-g , Changes of in vivo AML blast counts or AML blast ratio through the days after ABT-199 administration. The estimated scores and the PPI diagnostic results (R: Responsive, NR: Non-responsive) were obtained from the model in Fig. . ( a ) BC-7052, ( b ) BC-8634, ( c ) BC-8784, ( d ) BC-7081, ( e ) BC-9458, ( f ) BC-7064, ( g ) BC-7082. The BIM BH3 PBA profiles of each sample were presented, and The data were normalized to the BCL2-BIM BH3 PBA level of HL60 cell. h , Comparison of the estimated scores of AML samples between responsive (R, n = 4) and non-responsive (NR, n = 10) samples for in vivo ABT-199 administration (Two-sided Mann-Whitney test, p -value = 0.004). Six additional samples from the same cohort collected after relapse (BC-7064-R, BC-7082-R, BC-7107-R2, BC-8900, BC-9458, BC-9492) were included, which were categorized as the NR. The centre line represents the median, the box limits are upper and lower quartiles, and the whiskers represent 1.5x interquartile range for the box plots.

Article Snippet: All full-length BCL2-family proteins ( BCL2 (HG10195-M, SinoBiological), BCLxL (HG10455-M, SinoBiological), MCL1 (HG10240-M, SinoBiological), BIM EL (HG13816-G, SinoBiological), BAD (HG10020-M, SinoBiological), BAX (HG11619-M, SinoBiological), BAK (HG10450-M, SinoBiological) and NOXA (HG16548-U, SinoBiological)) were isolated from their respective human complementary (c)DNA. cDNA of BIM BH3 (MRQAEPADMRPEIWIAQELRRIGDEFNAYYARR) was isolated from BIM EL cDNA. cDNA of BAK fragments (residues 24–69 (α 1 helix), 70–91 (α 2 –α 3 helices), 90–150 (α 3 –α 5 helices) and 146–187 (α 6 –α 8 helices)) were isolated from BAK cDNA.

Techniques: In Vivo, Diagnostic Assay, Comparison, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Molecular interactions among tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), Beclin 1, and ubiquitin-specific protease 14 (USP14). (A) Truncated mutants of Beclin 1. Myc-tagged Beclin 1 truncated mutants were generated as described in Section “ .” BH3, Bcl-2 Homology 3; CCD, coiled coil domain; ECD, evolutionarily conserved domain. (B) Expression vectors of Myc-tagged Beclin 1 wild type (wt) and truncated mutants were co-transfected with Flag-tagged USP14 plasmid into HEK293T cells followed by immunoprecipitation (IP) and western blotting analyses. (C) Myc-tagged Beclin 1 wt, Myc-tagged Beclin 1 truncated mutants, and mock as control plasmid were co-transfected with Flag-tagged TRAF6 into HEK293T cells followed by IP and western blotting analyses. (D) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged Beclin 1 into HEK293T cells followed by IP and western blotting analyses. (E) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged USP14 into HEK293T cells followed by IP and western blotting analyses. (F) A schematic model showing molecular interactions among TRAF6, Beclin 1, and USP14. TRAF-C, C-terminal TRAF domain.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Generated, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) inhibits ubiquitination of Beclin 1. (A) Myc-tagged Beclin 1, Flag-tagged tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and mock as control plasmid were co-transfected with Flag-tagged USP14 plasmid at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to immuno-blotting (IB) assay using anti-USP14, anti-TRAF6, anti-Flag, or anti-Myc antibody. *The intensity of Flag-USP14 was measured by ImageJ. Fold change relative to Flag-USP14 of lane 3 was calculated. **The intensity of Flag-TRAF6 was measured by ImageJ. Fold change relative to Flag-TRAF6 of lane 2 was calculated. (B) Myc-tagged Beclin 1, HA-tagged Ub, Flag-tagged TRAF6, and mock as control plasmid were co-transfected with Flag-tagged USP14 vector at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to IB assay using anti-HA, anti-Myc, anti-USP14, or anti-TRAF6 antibody. (C) A schematic model showing the inhibition of Beclin 1 ubiquitination by USP14.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Inhibition

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) negatively regulates autophagy induction and toll-like receptor 4 (TLR4)-mediated signaling. (A) Upon TLR4 stimulation, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interacts with Beclin 1 through its coiled coil (CC) domain, induces ubiquitination of Beclin 1, and facilitate autophagy induction (upper). In contrast, USP14 competitively interacts with Beclin 1 to the CC domain of TRAF6. This leads to the suppression of Beclin 1 ubiquitination by TRAF6, resulting in the inhibition of autophagy induction (down). (B) Following TLR4 stimulation, ubiquitinated TRAF6 is associated with the TAB 2–TAK1–TAB 1 complex through an interaction between its polyubiquitinated chain and TAB 2. TAB 2 is then ubiquitinated and the complex facilitates the activation of TAK1. Simultaneously, the IKK complex is associated with the former complex through the polyubiquitinated chain and TAB 2, leading to the activations of nuclear factor-kappa B (NF-κB) and p38 (left). In contrast, the interaction between TAB 2 and USP14 may lead to the deubiquitination of TAB 2, thus inhibiting the association of IKKs, which inhibits the activation of NF-κB and p38 (right).

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Inhibition, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin

doi: 10.1073/pnas.2020478118

Figure Lengend Snippet: Whole-genome CRISPR/Cas9 screen to identify drivers of breast cancer cell proliferation. ( A and B ) Volcano plots of the CRISPR screen analyses demonstrating enriched sgRNAs (targeting the listed genes) for MCF7.control cells ( A ) and MCF7. beclin 1 cells ( B ) after 4 wk of cell proliferation. Genes targeted by the most significantly enriched sgRNAs are highlighted in red. The x -axis represents the average LFC of all the sgRNAs that target a gene, and the y -axis represents the average –log 10 P values for all the sgRNAs targeting a gene. P values were calculated using hypergeometric distribution. See also SI Appendix , Fig. S2 .

Article Snippet: After 24 h, cells were either mock transfected or transfected with a pBicep-3xFLAG-Beclin 1 vector using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: CRISPR