Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 1. FE65 enhances autophagic activity. (A) The overexpression of FE65 in stable cell lines increased the level of free GFP from the cleavage of GFP-LC3, with or without CQ treatment. (B) The knockout of FE65 in cells attenuated the release of free GFP from GFP-LC3, again with or without CQ treatment. GFP-LC3 and GFP were detected in Western blots using an anti-GFP antibody, while the expression of FE65 was confirmed with an anti-FE65 antibody (E20). (A,B) The bar charts show the levels of free GFP; error bars represent standard deviation (SD), *** p < 0.001. (C) FE65 overexpressing stable cells exhibited increased lipidation of LC3 when treated with or without CQ. Endogenous LC3 was detected by immunoblotting using an anti-LC3 (Proteintech) antibody. (D) WT HEK293 and FE65 KO cells were treated with or without CQ. The knockout of FE65 decreased the level of LC3-II. (C,D) The bar charts represent the levels of LC3-II; error bars are shown as SD, *** p < 0.001. (E) Stable

Article Snippet: Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65∆Ct. (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz).

Techniques: Activity Assay, Over Expression, Stable Transfection, Knock-Out, Western Blot, Expressing, Standard Deviation

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 2. FE65 is a novel Beclin 1 interactor. (A–D) FE65 interacts with Beclin 1. (A) Bacterial- expressed GST and GST-Beclin 1 were used as bait to pulldown FE65 in transfected cell lysates. FE65 was specifically pulled down by GST-Beclin 1, with the amount of bait used in each pulldown shown in a Coomassie blue-stained gel. (B) Upper panel: HEK293 cells were transfected with either FE65 alone or FLAG-Beclin 1. Beclin 1 was immunoprecipitated using (−) a control mouse IgG or (+) an anti- FLAG antibody (M2, Sigma). The co-immunoprecipitant was detected using an anti-FE65 antibody (E20, Santa Cruz). Lower panel: HEK293 cells were transfected with myc-FE65 + FLAG-ATG14L, myc-FE65 + FLAG-Beclin 1, myc-FE65 + FLAG-VPS15, and myc-FE65 + HA-VPS34. The PI3KC3- C1 components were immunoprecipitated using either (−) control mouse IgG or (+) anti-FLAG antibody (M2, Sigma) or anti-HA antibody (12CA5, Roche). The level of FE65 in immunoprecipitate was detected using an anti-myc antibody (9B11, Cell Signaling Technology). (C) FE65 interacts with Beclin 1 endogenously in rat brain lysate. Beclin 1 in rat brain lysate was immunoprecipitated using an

Article Snippet: Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65∆Ct. (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz).

Techniques: Transfection, Staining, Immunoprecipitation, Control

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 3. FE65-Beclin 1 interaction is essential to facilitate Beclin 1-mediated autophagy. (A) A GFP-LC3 cleavage assay was performed in WT HEK293 and FE65 KO cells, with and without CQ treatment. The knockout of FE65 decreased the level of free GFP from GFP-LC3 cleavage, and the overexpression of Beclin 1 failed to potentiate GFP-LC3 cleavage in FE65 KO cells. The bar chart shows the level of GFP; error bars represent standard deviation (SD), *** p < 0.001. (B) FE65, but not FE65∆Ct, increased endogenous LC3 lipidation in stably expressing cells, with and without Baf A1 treatment. The bar chart represents the level of LC3-II; error bars are shown as SD, *** p < 0.001, ** p < 0.01. (C) FE65-stably transfected cells showed a decrease in the p62/Beclin 1 ratio with and without Baf A1 treatment, whereas FE65∆Ct did not exhibit this decrease. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. *** p < 0.001, ** p < 0.01, * p < 0.05. (D,E) FE65 and FE65∆Ct were transfected in FE65 KO cells to assess autophagic activity. (D) Only FE65 was able to rescue the lipidation of LC3, regardless of CQ treatment. The bar chart illustrates the level of LC3-II; error bars are shown as SD, ** p < 0.01, * p < 0.05. (E) FE65 transfection in FE65 KO cells led to a significant decrease in the p62/Beclin 1 ratio, both with and without CQ treatment, while transfection with FE65∆Ct attenuated this effect. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. ** p < 0.01, * p < 0.05. (F) FE65 and FE65∆Ct were transfected into GFP-LC3 stably expressing cells, with and without Baf A1 treatment.

Article Snippet: Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65∆Ct. (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz).

Techniques: Cleavage Assay, Knock-Out, Over Expression, Standard Deviation, Stable Transfection, Expressing, Transfection, Activity Assay

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 4. FE65 regulates autophagy by promoting PI3KC3-C1 activity. (A) The knockout of FE65 decreased the kinase activity of PI3KC3-C1, as indicated by a reduction in the production of PI3P.

Article Snippet: Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65∆Ct. (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz).

Techniques: Activity Assay, Knock-Out

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 2. FE65 is a novel Beclin 1 interactor. (A–D) FE65 interacts with Beclin 1. (A) Bacterial- expressed GST and GST-Beclin 1 were used as bait to pulldown FE65 in transfected cell lysates. FE65 was specifically pulled down by GST-Beclin 1, with the amount of bait used in each pulldown shown in a Coomassie blue-stained gel. (B) Upper panel: HEK293 cells were transfected with either FE65 alone or FLAG-Beclin 1. Beclin 1 was immunoprecipitated using (−) a control mouse IgG or (+) an anti- FLAG antibody (M2, Sigma). The co-immunoprecipitant was detected using an anti-FE65 antibody (E20, Santa Cruz). Lower panel: HEK293 cells were transfected with myc-FE65 + FLAG-ATG14L, myc-FE65 + FLAG-Beclin 1, myc-FE65 + FLAG-VPS15, and myc-FE65 + HA-VPS34. The PI3KC3- C1 components were immunoprecipitated using either (−) control mouse IgG or (+) anti-FLAG antibody (M2, Sigma) or anti-HA antibody (12CA5, Roche). The level of FE65 in immunoprecipitate was detected using an anti-myc antibody (9B11, Cell Signaling Technology). (C) FE65 interacts with Beclin 1 endogenously in rat brain lysate. Beclin 1 in rat brain lysate was immunoprecipitated using an

Article Snippet: FLAG-Beclin 1 was immunoprecipitated with (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma), and the immunoprecipitant of FE65 was detected using an anti-myc antibody (Proteintech).

Techniques: Transfection, Staining, Immunoprecipitation, Control

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65 enhances autophagic activity. ( A ) The overexpression of FE65 in stable cell lines increased the level of free GFP from the cleavage of GFP-LC3, with or without CQ treatment. ( B ) The knockout of FE65 in cells attenuated the release of free GFP from GFP-LC3, again with or without CQ treatment. GFP-LC3 and GFP were detected in Western blots using an anti-GFP antibody, while the expression of FE65 was confirmed with an anti-FE65 antibody (E20). ( A , B ) The bar charts show the levels of free GFP; error bars represent standard deviation (SD), *** p < 0.001. ( C ) FE65 overexpressing stable cells exhibited increased lipidation of LC3 when treated with or without CQ. Endogenous LC3 was detected by immunoblotting using an anti-LC3 (Proteintech) antibody. ( D ) WT HEK293 and FE65 KO cells were treated with or without CQ. The knockout of FE65 decreased the level of LC3-II. ( C , D ) The bar charts represent the levels of LC3-II; error bars are shown as SD, *** p < 0.001. ( E ) Stable cells overexpressing FE65 showed a decrease in the p62/Beclin 1 ratio, regardless of CQ treatment. Endogenous p62 was detected by immunoblotting using an anti-p62 (Thermofisher) antibody. ( F ) WT HEK293 and FE65 KO cells were treated with or without CQ. The knockout of FE65 resulted in increased p62 accumulation. ( E , F ) The bar charts illustrate the ratio of p62 to Beclin 1, with error bars representing SD, *** p < 0.001, ** p < 0.01. ( G ) GFP-LC3 stably expressing cells were transfected with control and FE65 siRNA. GFP-LC3 puncta were counted after starvation treatment with serum-free medium for 3 h. The bar chart shows the number of puncta per cell. Data were obtained from at least 70 cells per transfection, and the experiment was repeated at least three times. Error bars are SEM, *** p < 0.001. Scale bar, 10 μm. Western blotting also confirmed the knockdown of FE65 in the stable cell line.

Article Snippet: After blocking in 5% FBS, cells were incubated with goat anti-FE65 antibody (E20, Santa Cruz) [1:2000] and mouse anti-FLAG antibody (M2, Sigma) [1:2000] for 1 h at room temperature. β-tubulin and the nucleus were also stained by anti-β-tubulin and DAPI, respectively, to monitor the cell morphology.

Techniques: Activity Assay, Over Expression, Stable Transfection, Knock-Out, Western Blot, Expressing, Standard Deviation, Transfection, Control, Knockdown

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65 is a novel Beclin 1 interactor. ( A – D ) FE65 interacts with Beclin 1. ( A ) Bacterial-expressed GST and GST-Beclin 1 were used as bait to pulldown FE65 in transfected cell lysates. FE65 was specifically pulled down by GST-Beclin 1, with the amount of bait used in each pulldown shown in a Coomassie blue-stained gel. ( B ) Upper panel: HEK293 cells were transfected with either FE65 alone or FLAG-Beclin 1. Beclin 1 was immunoprecipitated using (−) a control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma). The co-immunoprecipitant was detected using an anti-FE65 antibody (E20, Santa Cruz). Lower panel: HEK293 cells were transfected with myc-FE65 + FLAG-ATG14L, myc-FE65 + FLAG-Beclin 1, myc-FE65 + FLAG-VPS15, and myc-FE65 + HA-VPS34. The PI3KC3-C1 components were immunoprecipitated using either (−) control mouse IgG or (+) anti-FLAG antibody (M2, Sigma) or anti-HA antibody (12CA5, Roche). The level of FE65 in immunoprecipitate was detected using an anti-myc antibody (9B11, Cell Signaling Technology). ( C ) FE65 interacts with Beclin 1 endogenously in rat brain lysate. Beclin 1 in rat brain lysate was immunoprecipitated using an anti-Beclin 1 antibody (Proteintech), and the co-immunoprecipitant of FE65 was detected by an anti-FE65 antibody (E20, Santa Cruz). ( D ) The interaction of FE65 and Beclin 1 was confirmed in cellulo by proximity ligation assay (PLA). HEK293 cells were transfected with FE65 or FE65 ΔCt alone, FLAG-Beclin 1 alone, and FE65 or FE65 ΔCt + FLAG-Beclin 1. Goat anti-FE65 antibody (E20, Santa Cruz) [1:2000] and mouse anti-FLAG antibody (M2, Sigma) [1:2000] were used. Cells co-transfected with both FE65 and Beclin 1 exhibited an increase in PLA signals, demonstrating the interaction of FE65-Beclin1 in cellulo. PLA signals were decreased significantly in FE65 ΔCt + FLAG-Beclin 1 co-transfected cells, indicating an impaired interaction. The bar chart shows the PLA signals per cell. Data were obtained from at least 40 cells per transfection. The experiment was repeated at least three times. Error bars represent SEM, *** p < 0.001. Scale bar, 10 μm. ( E ) Immunostaining images of COS7 cells for FLAG-Beclin 1 and myc-FE65. FLAG-Beclin 1 and myc-FE65 were stained with anti-FLAG antibody (M2, Sigma) and anti-FE65 antiserum (rabbit), respectively. Nuclei were stained with DAPI. An overlapped image is shown. Scale bar, 10 μm. ( F ) The C-terminal region of FE65, containing PTB2 and the C-terminus, is responsible for interacting with Beclin 1. Constructs of different FE65 deletion mutants were generated, as shown in the upper schematic diagrams. FE65 deletion mutants were co-transfected with FLAG-Beclin 1 in HEK293 cells. FLAG-Beclin 1 was immunoprecipitated with (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma), and the immunoprecipitant of FE65 was detected using an anti-myc antibody (Proteintech). Only the deletion of PTB2 and the C-terminus was able to disrupt the FE65-Beclin 1 interaction. ( G ) FE65 ΔCt significantly impaired the FE65-Beclin 1 interaction. Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65 ΔCt . (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz). The level of FE65 that was immunoprecipitated is shown as a bar chart; error bars represent SD, *** p < 0.001. ( H ) Beclin 1 350–400 is responsible for interacting with FE65. GST-tagged constructs of Beclin 1 fragments were generated, as shown in the upper schematic diagrams. GST-Beclin 1 fragments were used as bait to pulldown FE65 in transfected cell lysates. All Beclin 1 fragments containing amino acids 350–400 were able to pulldown FE65; only the fragment Beclin 1 400–450 showed an inability to pull down FE65, indicating that the Beclin 1 350–400 fragment is responsible for the interaction with FE65. In ( A – C , E – G ), 1% of cell/tissue lysates were loaded as size controls.

Article Snippet: After blocking in 5% FBS, cells were incubated with goat anti-FE65 antibody (E20, Santa Cruz) [1:2000] and mouse anti-FLAG antibody (M2, Sigma) [1:2000] for 1 h at room temperature. β-tubulin and the nucleus were also stained by anti-β-tubulin and DAPI, respectively, to monitor the cell morphology.

Techniques: Transfection, Staining, Immunoprecipitation, Control, Proximity Ligation Assay, Immunostaining, Construct, Generated

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65-Beclin 1 interaction is essential to facilitate Beclin 1-mediated autophagy. ( A ) A GFP-LC3 cleavage assay was performed in WT HEK293 and FE65 KO cells, with and without CQ treatment. The knockout of FE65 decreased the level of free GFP from GFP-LC3 cleavage, and the overexpression of Beclin 1 failed to potentiate GFP-LC3 cleavage in FE65 KO cells. The bar chart shows the level of GFP; error bars represent standard deviation (SD), *** p < 0.001. ( B ) FE65, but not FE65 ΔCt , increased endogenous LC3 lipidation in stably expressing cells, with and without Baf A1 treatment. The bar chart represents the level of LC3-II; error bars are shown as SD, *** p < 0.001, ** p < 0.01. ( C ) FE65-stably transfected cells showed a decrease in the p62/Beclin 1 ratio with and without Baf A1 treatment, whereas FE65 ΔCt did not exhibit this decrease. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. *** p < 0.001, ** p < 0.01, * p < 0.05. ( D , E ) FE65 and FE65 ΔCt were transfected in FE65 KO cells to assess autophagic activity. ( D ) Only FE65 was able to rescue the lipidation of LC3, regardless of CQ treatment. The bar chart illustrates the level of LC3-II; error bars are shown as SD, ** p < 0.01, * p < 0.05. ( E ) FE65 transfection in FE65 KO cells led to a significant decrease in the p62/Beclin 1 ratio, both with and without CQ treatment, while transfection with FE65 ΔCt attenuated this effect. The bar chart represents the ratio of p62/Beclin 1, with error bars indicating SD. ** p < 0.01, * p < 0.05. ( F ) FE65 and FE65 ΔCt were transfected into GFP-LC3 stably expressing cells, with and without Baf A1 treatment. GFP-LC3 puncta were counted, and overexpression of FE65 increased the number of puncta per cell, whereas overexpression of FE65 ΔCt failed to increase GFP-LC3 puncta. The bar chart shows the average number of puncta in cells; data were obtained from at least 40 cells, and the experiments were repeated at least 3 times. Error bars are SEM, *** p < 0.001, ** p < 0.01. Scale bar, 10 μm.

Article Snippet: After blocking in 5% FBS, cells were incubated with goat anti-FE65 antibody (E20, Santa Cruz) [1:2000] and mouse anti-FLAG antibody (M2, Sigma) [1:2000] for 1 h at room temperature. β-tubulin and the nucleus were also stained by anti-β-tubulin and DAPI, respectively, to monitor the cell morphology.

Techniques: Cleavage Assay, Knock-Out, Over Expression, Standard Deviation, Stable Transfection, Expressing, Transfection, Activity Assay

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65

doi: 10.3390/biology14010097

Figure Lengend Snippet: FE65 regulates autophagy by promoting PI3KC3-C1 activity. ( A ) The knockout of FE65 decreased the kinase activity of PI3KC3-C1, as indicated by a reduction in the production of PI3P. ( B ) Overexpression of FE65, but not FE65 ΔCt , increased PI3KC3-C1 kinase activity. ( A , B ) The core subunits of PI3KC3-C1 were transfected into cells, and the complex was isolated using an anti-myc antibody (Proteintech). The isolated complex was incubated with PI lipid substrate and ATP. The level of PI3P was detected using dot blot analysis. The bar chart shows the level of PI3P; error bars are shown as SD, ** p < 0.01, * p < 0.05. ( C ) Immunostaining of WIPI-2 in WT HEK293 and FE65 KO cells, with and without EBSS treatment. WIPI-2 puncta were counted; the knockout of FE65 in cells has decreased the number of WIPI-2 puncta per cell. ( D ) Immunostaining of WIPI-2 in FE65 and FE65 ΔCt transfected cells, with and without EBSS treatment. The transient overexpression of FE65 has significantly increased the WIPI-2 puncta formation in cells, while the transient overexpression of FE65 ΔCt failed to do so. ( C , D ) The bar chart shows the average number of puncta in cells; data were obtained from at least 40 cells, and the experiments were repeated at least 3 times. Error bars are SEM, *** p < 0.001, ** p < 0.01, * p < 0.05. Scale bar, 10 μm.

Article Snippet: After blocking in 5% FBS, cells were incubated with goat anti-FE65 antibody (E20, Santa Cruz) [1:2000] and mouse anti-FLAG antibody (M2, Sigma) [1:2000] for 1 h at room temperature. β-tubulin and the nucleus were also stained by anti-β-tubulin and DAPI, respectively, to monitor the cell morphology.

Techniques: Activity Assay, Knock-Out, Over Expression, Transfection, Isolation, Incubation, Dot Blot, Immunostaining

Journal: Biology

Article Title: Beclin 1-Mediated Autophagy Is Potentiated by an Interaction with the Neuronal Adaptor FE65.

doi: 10.3390/biology14010097

Figure Lengend Snippet: Figure 2. FE65 is a novel Beclin 1 interactor. (A–D) FE65 interacts with Beclin 1. (A) Bacterial- expressed GST and GST-Beclin 1 were used as bait to pulldown FE65 in transfected cell lysates. FE65 was specifically pulled down by GST-Beclin 1, with the amount of bait used in each pulldown shown in a Coomassie blue-stained gel. (B) Upper panel: HEK293 cells were transfected with either FE65 alone or FLAG-Beclin 1. Beclin 1 was immunoprecipitated using (−) a control mouse IgG or (+) an anti- FLAG antibody (M2, Sigma). The co-immunoprecipitant was detected using an anti-FE65 antibody (E20, Santa Cruz). Lower panel: HEK293 cells were transfected with myc-FE65 + FLAG-ATG14L, myc-FE65 + FLAG-Beclin 1, myc-FE65 + FLAG-VPS15, and myc-FE65 + HA-VPS34. The PI3KC3- C1 components were immunoprecipitated using either (−) control mouse IgG or (+) anti-FLAG antibody (M2, Sigma) or anti-HA antibody (12CA5, Roche). The level of FE65 in immunoprecipitate was detected using an anti-myc antibody (9B11, Cell Signaling Technology). (C) FE65 interacts with Beclin 1 endogenously in rat brain lysate. Beclin 1 in rat brain lysate was immunoprecipitated using an

Article Snippet: Co-immunoprecipitation assays were performed with FLAG-Beclin 1 co-transfected with either FE65 or FE65∆Ct. (−) control mouse IgG or (+) an anti-FLAG antibody (M2, Sigma) was used to immunoprecipitate FLAG-Beclin 1, and the level of FE65 in the immunoprecipitate was detected using an anti-FE65 antibody (E20, Santa Cruz).

Techniques: Transfection, Staining, Immunoprecipitation, Control