human colonic epithelial cell line ccd841con  (ATCC)

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: Loss of ABHD5 suppresses CASP-independent cell death induced by nutrition deprivation. (A) GSEA plot of autophagy, apoptosis and WNT signaling pathways between ABHD5 high and ABHD5 low subgroups. (B) A heatmap of pathway enrichment signature in ABHD5 high and low subgroups. (C) ABHD5+/+ (WT) and ABHD5−/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (D) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells colonies were exposed to EBSS culture, and the colony survival was calculated at the indicated time points by crystal violet and trypan blue exclusion assay. (E) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS for 0, 3, 6 or 12 h , and the cell viability was determined by MTT assay. (F) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells stably transfected with a wild-type ABHD5 expression plasmid or control empty vector were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (G) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (H) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS for 3 h and analyzed by transmission electron microscopy. Arrows, autophagosomes; M, mitochondria; LD, lipid droplet; N, nucleus. The number of autophagosomes per cross-sectioned cell was counted. (I) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were infected with GFP-RFP-LC3 adenovirus; 24 h after infection, high-content screen images showing RFP- and GFP-labeled LC3 staining in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at different time points (2, 4, 8 and 16 h in the presence of EBSS culture). Scale bar: 5 μm. Statistical analysis showing autophagosomes, autolysosomes and the corresponding cell viability (J) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at the indicated timepoints. (K) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at 24 h following exposure to PBS, EBSS or EBSS+rapamycin (100 nM). (L) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS in the presence of rapamycin (Rap, 100nM), dihydro-N-acetyl-d-erythro-sphingosine (NADS, 10 mM), brefeldin A (Bre A, 5 µM) or control PBS for 0, 3, 6, 12 or 18 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. Unless noted, all bar plots in the figure are mean ± SEM of n biological replicates. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Protein-Protein interactions, Cell Culture, Trypan Blue Exclusion Assay, MTT Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, Transmission Assay, Electron Microscopy, Infection, Labeling, Staining, Western Blot

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: Loss of ABHD5 promotes DNA damage and genome instability in an autophagy-dependent manner. (A) ABHD5+/+ (WT) and ABHD5−/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 12 h, and the DNA damage was measured by alkaline single cell gel electrophoresis (Comet) assay. Error bars of statistical analysis of tail length represent standard error. (B) Immunostaining of γH2AFX (green) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at the indicated time points after exposure to EBSS. DAPI was used to stain the nuclei. (C) Quantification of γH2AFX-positive foci from (B). (D) Western blots of DNA damage-associated proteins in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells 24 h following exposure to EBSS. (E) Representative photomicrographs of ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells stained by indirect immunofluorescence for microtubules (anti-TUBULIN), DNA (DAPI), and centrosome-related protein (anti-NDEL1/NUDEL). Note the extensive microtubule network, the heterogeneous nuclear size, and the abnormalities in NDEL1 expression in ABHD5−/− (KO) cells. (F) Quantification of centrosome numbers in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells. Percentage of cells with normal centrosome numbers (one or 2; blue bar) and with supernumerary centrosomes (more than 2; red bar). (G) Flow cytometry analysis of a panel of ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells showing their DNA content. (H) Analysis of total DNA content and aneuploid DNA content in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells. (I) Statistical analysis of tail length, γH2AFX-positive foci and centrosome numbers in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells matched with or without rapamycin treatment (50 nmol/L, 24 h). Rapamycin treatment significantly reversed the tail length, γH2AFX-positive foci and centrosome numbers in ABHD5−/− (KO) cells to the level of control cells. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Cell Culture, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Immunostaining, Staining, Western Blot, Immunofluorescence, Expressing, Flow Cytometry, Control

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 regulates autophagic flux independent of its canonical metabolic activity. (A) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5-silenced, PNPLA2-silenced and control CCD841CON cells 24 h following exposure to EBSS. (B) PNPLA2-silenced and control CCD841CON cells were treated with EBSS for 24 h, and the cell viability was determined by MTT assay. (C) Western blots of PNPLA2 in control and CRISPR/Cas9-mediated PNPLA2 knockout (KO) CCD841CON cells. (D) ABHD5 expression was silenced in PNPLA2 knockout CCD841CON cells, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (E) Predicted domain structure of ABHD5, including the α/β hydrolase domain. The sites of mutations are indicated. (F) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were transfected with mutated ABHD5 (Q130P and E260K) or control vector plasmids. Statistical analysis of the number of autophagosomes/cell in different groups (a random number of 30 cells were selected for each group). (G) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were transfected with mutated ABHD5 (Q130P and E260K) or control vector plasmids. Cell viability of cells at different time points (6, 12, 24, 48, 72 h) after the exposure to EBSS was measured by MTT assay. (**, p < 0.001; ns, no significance).

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Activity Assay, Western Blot, Control, MTT Assay, CRISPR, Knock-Out, Expressing, Trypan Blue Exclusion Assay, Transfection, Plasmid Preparation

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 promotes autophagy via protecting BECN1 from being cleaved. (A) Representative immunofluorescent images of staining with the indicated antibodies in wild-type CCD841CON cells are shown. LC3 and BECN1 proteins were labeled with green-fluorescent Alexa Fluor 488, ABHD5 were visualized with red-fluorescent Alexa Fluor 594 and nuclei with DAPI (blue). An overlay is presented in the ‘Merge’ panel. (B) Immunoprecipitation of endogenous ABHD5 with LC3 or BECN1 in ABHD5-silenced and control CCD841CON cells. (C) Whole-cell lysates were prepared from ABHD5-silenced and control CCD841CON cells matched with or without EBSS (24 h) treatment, and analyzed by western blotting with anti-BECN1 antibody. (D) EBSS pretreated ABHD5-silenced and control CCD841CON cells were subjected to subcellular fractionation. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control. (E) EBSS-pretreated ABHD5-silenced and control CCD841CON cells were subjected to subcellular fractionation. The nucleic fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (F) EBSS-pretreated ABHD5-silenced and control CCD841CON cells were subjected to immunofluorescent staining with BECN1-C-specific antibody labeled with Alexa Fluor 488 (green) and MitoTracker (red, a marker of mitochondria) to reveal the localization of BECN1-C on mitochondria (orange), and immunofluorescent staining with BECN1-N-specific antibody labeled with Alexa Fluor 594 (red) and MitoTracker (green) to reveal the localization of BECN1-N in the nucleus (purple). (G) ABHD5-silenced and control CCD841CON cells were transfected with full-length BECN1 or BECN1-C and subjected to EBSS treatment. Number of autolysosomes and cell viability were statistically analyzed. (H) ApcMin/− Abhd5+/+/Cre+ (control mice) and ApcMin/− Abhd5f/f/Cre+ (intestinal Abhd5 knockout ApcMin/−) mice were intraperitoneally administered with a specific activator of BECN1 (1.5 mg/kg, once daily from the age of 30 d until the age of 100 d), and the tumor number and size in the entire small intestine were statistically analyzed. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Staining, Labeling, Immunoprecipitation, Control, Western Blot, Fractionation, Marker, Transfection, Knock-Out

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 directly interacts with BECN1 to inhibit the cleavage of BECN1. (A) Reciprocal GST affinity isolation and immunoblotting of ABHD5 and BECN1 in wild-type CCD841CON cells. (B) GST-tagged ABHD5 was purified and incubated with increasing doses of purified BECN1 for in vitro detection of their direct association. (C) Immunoblotting of ABHD5 in the lysates of CCD841CON cells affinity isolated with GST-tagged full-length or different domains of BECN1 as indicated. The domain structure of BECN1 is given above the data. (D) In vitro binding assay with FLAG-tagged different domains of ABHD5 and His-tagged full-length BECN1 as indicated. The domain structure of ABHD5 is given above the data. (E) A close-up view of the ionic layer at the center of the crystal structure of the ABHD5-BECN1 complex is shown. (F) ABHD5-silenced and control cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (G, H) ABHD5-silenced and control CCD841CON cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. Number of autolysosomes and cell viability were statistically analyzed.(*, p < 0.01; ns, no significance).

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Isolation, Western Blot, Purification, Incubation, In Vitro, Binding Assay, Control, Transfection, Mutagenesis

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 competitively occupies the CASP3-dependent cleavage sites of BECN1 to protect BECN1 from being cleaved by CASP3. (A) Western blots of apoptosis-related proteins in whole ABHD5-silenced and control CCD841CON cells 24 h after treatment with PBS, EBSS or EBSS+rapamycin (50 nM). (B) CRISPR/Cas9-mediated CASP3 knockout (KO) CCD841CON cells were subjected to ABHD5 knockdown and a follow-up challenge of EBSS. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (C) A scheme representing BECN1. Sequence alignment of human BECN1 shows conservation of the CASP3 cleavage sites, DLFD124, TDVD133 and DQLD149. The CASP3-dependent cleavage sites are indicated in bold. The BH3, coiled-coil domain (CCD) and evolutionarily conserved domain (ECD) are depicted. (D) In vitro binding assay with FLAG-tagged different domains of BECN1 and His-tagged CASP3 or ABHD5 as indicated. The indicated A, B or C domain structure of BECN1 is given in (C). (E) Representative immunofluorescent images of staining with the indicated antibodies are shown. ABHD5, BECN1 and CASP3 are shown as blue, red, and green, respectively. An overlay (purple) in control cells reveals an interaction between ABHD5 and BECN1, and an overlay (orange) in ABHD5-silenced cells reveals an interaction between BECN1 and CASP3. (F) Immunoprecipitation of endogenous BECN1 with LC3 in ABHD5-silenced and control CCD841CON cells.

Article Snippet: Cell culture Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Western Blot, Control, CRISPR, Knock-Out, Knockdown, Sequencing, In Vitro, Binding Assay, Staining, Immunoprecipitation

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: Loss of ABHD5 suppresses CASP-independent cell death induced by nutrition deprivation. (A) GSEA plot of autophagy, apoptosis and WNT signaling pathways between ABHD5 high and ABHD5 low subgroups. (B) A heatmap of pathway enrichment signature in ABHD5 high and low subgroups. (C) ABHD5+/+ (WT) and ABHD5−/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (D) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells colonies were exposed to EBSS culture, and the colony survival was calculated at the indicated time points by crystal violet and trypan blue exclusion assay. (E) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS for 0, 3, 6 or 12 h , and the cell viability was determined by MTT assay. (F) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells stably transfected with a wild-type ABHD5 expression plasmid or control empty vector were cultured in EBSS for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (G) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS in the presence of 50 μM z-VAD-fmk or control DMSO for 0, 3, 6 or 12 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (H) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS for 3 h and analyzed by transmission electron microscopy. Arrows, autophagosomes; M, mitochondria; LD, lipid droplet; N, nucleus. The number of autophagosomes per cross-sectioned cell was counted. (I) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were infected with GFP-RFP-LC3 adenovirus; 24 h after infection, high-content screen images showing RFP- and GFP-labeled LC3 staining in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at different time points (2, 4, 8 and 16 h in the presence of EBSS culture). Scale bar: 5 μm. Statistical analysis showing autophagosomes, autolysosomes and the corresponding cell viability (J) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at the indicated timepoints. (K) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at 24 h following exposure to PBS, EBSS or EBSS+rapamycin (100 nM). (L) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were cultured in EBSS in the presence of rapamycin (Rap, 100nM), dihydro-N-acetyl-d-erythro-sphingosine (NADS, 10 mM), brefeldin A (Bre A, 5 µM) or control PBS for 0, 3, 6, 12 or 18 h, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. Unless noted, all bar plots in the figure are mean ± SEM of n biological replicates. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Protein-Protein interactions, Cell Culture, Trypan Blue Exclusion Assay, MTT Assay, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, Transmission Assay, Electron Microscopy, Infection, Labeling, Staining, Western Blot

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: Loss of ABHD5 promotes DNA damage and genome instability in an autophagy-dependent manner. (A) ABHD5+/+ (WT) and ABHD5−/− (KO) colon epithelial cells (CCD841CON) were cultured in EBSS for 12 h, and the DNA damage was measured by alkaline single cell gel electrophoresis (Comet) assay. Error bars of statistical analysis of tail length represent standard error. (B) Immunostaining of γH2AFX (green) in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells at the indicated time points after exposure to EBSS. DAPI was used to stain the nuclei. (C) Quantification of γH2AFX-positive foci from (B). (D) Western blots of DNA damage-associated proteins in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells 24 h following exposure to EBSS. (E) Representative photomicrographs of ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells stained by indirect immunofluorescence for microtubules (anti-TUBULIN), DNA (DAPI), and centrosome-related protein (anti-NDEL1/NUDEL). Note the extensive microtubule network, the heterogeneous nuclear size, and the abnormalities in NDEL1 expression in ABHD5−/− (KO) cells. (F) Quantification of centrosome numbers in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells. Percentage of cells with normal centrosome numbers (one or 2; blue bar) and with supernumerary centrosomes (more than 2; red bar). (G) Flow cytometry analysis of a panel of ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells showing their DNA content. (H) Analysis of total DNA content and aneuploid DNA content in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells. (I) Statistical analysis of tail length, γH2AFX-positive foci and centrosome numbers in ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells matched with or without rapamycin treatment (50 nmol/L, 24 h). Rapamycin treatment significantly reversed the tail length, γH2AFX-positive foci and centrosome numbers in ABHD5−/− (KO) cells to the level of control cells. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Cell Culture, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Immunostaining, Staining, Western Blot, Immunofluorescence, Expressing, Flow Cytometry, Control

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 regulates autophagic flux independent of its canonical metabolic activity. (A) Western blots of autophagy-related proteins (LC3-I, LC3-II, SQSTM1) in ABHD5-silenced, PNPLA2-silenced and control CCD841CON cells 24 h following exposure to EBSS. (B) PNPLA2-silenced and control CCD841CON cells were treated with EBSS for 24 h, and the cell viability was determined by MTT assay. (C) Western blots of PNPLA2 in control and CRISPR/Cas9-mediated PNPLA2 knockout (KO) CCD841CON cells. (D) ABHD5 expression was silenced in PNPLA2 knockout CCD841CON cells, and the percentage of dead cells was determined at the indicated time points by trypan blue exclusion assay. (E) Predicted domain structure of ABHD5, including the α/β hydrolase domain. The sites of mutations are indicated. (F) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were transfected with mutated ABHD5 (Q130P and E260K) or control vector plasmids. Statistical analysis of the number of autophagosomes/cell in different groups (a random number of 30 cells were selected for each group). (G) ABHD5+/+ (WT) and ABHD5−/− (KO) CCD841CON cells were transfected with mutated ABHD5 (Q130P and E260K) or control vector plasmids. Cell viability of cells at different time points (6, 12, 24, 48, 72 h) after the exposure to EBSS was measured by MTT assay. (**, p < 0.001; ns, no significance).

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Activity Assay, Western Blot, Control, MTT Assay, CRISPR, Knock-Out, Expressing, Trypan Blue Exclusion Assay, Transfection, Plasmid Preparation

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 promotes autophagy via protecting BECN1 from being cleaved. (A) Representative immunofluorescent images of staining with the indicated antibodies in wild-type CCD841CON cells are shown. LC3 and BECN1 proteins were labeled with green-fluorescent Alexa Fluor 488, ABHD5 were visualized with red-fluorescent Alexa Fluor 594 and nuclei with DAPI (blue). An overlay is presented in the ‘Merge’ panel. (B) Immunoprecipitation of endogenous ABHD5 with LC3 or BECN1 in ABHD5-silenced and control CCD841CON cells. (C) Whole-cell lysates were prepared from ABHD5-silenced and control CCD841CON cells matched with or without EBSS (24 h) treatment, and analyzed by western blotting with anti-BECN1 antibody. (D) EBSS pretreated ABHD5-silenced and control CCD841CON cells were subjected to subcellular fractionation. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control. (E) EBSS-pretreated ABHD5-silenced and control CCD841CON cells were subjected to subcellular fractionation. The nucleic fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (F) EBSS-pretreated ABHD5-silenced and control CCD841CON cells were subjected to immunofluorescent staining with BECN1-C-specific antibody labeled with Alexa Fluor 488 (green) and MitoTracker (red, a marker of mitochondria) to reveal the localization of BECN1-C on mitochondria (orange), and immunofluorescent staining with BECN1-N-specific antibody labeled with Alexa Fluor 594 (red) and MitoTracker (green) to reveal the localization of BECN1-N in the nucleus (purple). (G) ABHD5-silenced and control CCD841CON cells were transfected with full-length BECN1 or BECN1-C and subjected to EBSS treatment. Number of autolysosomes and cell viability were statistically analyzed. (H) ApcMin/− Abhd5+/+/Cre+ (control mice) and ApcMin/− Abhd5f/f/Cre+ (intestinal Abhd5 knockout ApcMin/−) mice were intraperitoneally administered with a specific activator of BECN1 (1.5 mg/kg, once daily from the age of 30 d until the age of 100 d), and the tumor number and size in the entire small intestine were statistically analyzed. (*, p < 0.01; **, p < 0.001; ns, no significance).

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Staining, Labeling, Immunoprecipitation, Control, Western Blot, Fractionation, Marker, Transfection, Knock-Out

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 directly interacts with BECN1 to inhibit the cleavage of BECN1. (A) Reciprocal GST affinity isolation and immunoblotting of ABHD5 and BECN1 in wild-type CCD841CON cells. (B) GST-tagged ABHD5 was purified and incubated with increasing doses of purified BECN1 for in vitro detection of their direct association. (C) Immunoblotting of ABHD5 in the lysates of CCD841CON cells affinity isolated with GST-tagged full-length or different domains of BECN1 as indicated. The domain structure of BECN1 is given above the data. (D) In vitro binding assay with FLAG-tagged different domains of ABHD5 and His-tagged full-length BECN1 as indicated. The domain structure of ABHD5 is given above the data. (E) A close-up view of the ionic layer at the center of the crystal structure of the ABHD5-BECN1 complex is shown. (F) ABHD5-silenced and control cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (G, H) ABHD5-silenced and control CCD841CON cells were transfected with full-length ABHD5 or an ABHD5 mutant (mutated in the binding domain to BECN1) and subjected to EBSS treatment. Number of autolysosomes and cell viability were statistically analyzed.(*, p < 0.01; ns, no significance).

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Isolation, Western Blot, Purification, Incubation, In Vitro, Binding Assay, Control, Transfection, Mutagenesis

Journal: Autophagy

Article Title: ABHD5 interacts with BECN1 to regulate autophagy and tumorigenesis of colon cancer independent of PNPLA2

doi: 10.1080/15548627.2016.1217380

Figure Lengend Snippet: ABHD5 competitively occupies the CASP3-dependent cleavage sites of BECN1 to protect BECN1 from being cleaved by CASP3. (A) Western blots of apoptosis-related proteins in whole ABHD5-silenced and control CCD841CON cells 24 h after treatment with PBS, EBSS or EBSS+rapamycin (50 nM). (B) CRISPR/Cas9-mediated CASP3 knockout (KO) CCD841CON cells were subjected to ABHD5 knockdown and a follow-up challenge of EBSS. The cytosolic and mitochondrial fractions were analyzed by western blotting using anti-BECN1-C with COX4I as a loading control, and the nuclear fractions were analyzed by western blotting using anti-BECN1-N with histone 2A as a loading control. (C) A scheme representing BECN1. Sequence alignment of human BECN1 shows conservation of the CASP3 cleavage sites, DLFD124, TDVD133 and DQLD149. The CASP3-dependent cleavage sites are indicated in bold. The BH3, coiled-coil domain (CCD) and evolutionarily conserved domain (ECD) are depicted. (D) In vitro binding assay with FLAG-tagged different domains of BECN1 and His-tagged CASP3 or ABHD5 as indicated. The indicated A, B or C domain structure of BECN1 is given in (C). (E) Representative immunofluorescent images of staining with the indicated antibodies are shown. ABHD5, BECN1 and CASP3 are shown as blue, red, and green, respectively. An overlay (purple) in control cells reveals an interaction between ABHD5 and BECN1, and an overlay (orange) in ABHD5-silenced cells reveals an interaction between BECN1 and CASP3. (F) Immunoprecipitation of endogenous BECN1 with LC3 in ABHD5-silenced and control CCD841CON cells.

Article Snippet: Human colon epithelial cell lines CCD841CON (CRL-1790) and FHC (CRL-1831) were purchased from ATCC.

Techniques: Western Blot, Control, CRISPR, Knock-Out, Knockdown, Sequencing, In Vitro, Binding Assay, Staining, Immunoprecipitation