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tfeb-gfp plasmid (Addgene inc)

Name: tfeb-gfp plasmid
Brand: Addgene inc
Rating: 90 (1 reviews)

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Addgene inc tfeb-gfp plasmid
Tfeb Gfp Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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tfeb-gfp plasmid - by Bioz Stars, 2026-03

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C9orf72 -linked poly-GR and poly-PR inhibit the formation of autophagosomes under starvation condition. (A, B) HEK 293 cells were transfected with 0.1, 0.5 or 3.5 μg GFP-GR∗30, GFP-PR∗30 or GFP tag. After 48 h, the cells were incubated with Earle's balanced salt solution (EBSS) for 1 h, and cell lysates were subjected to immunoblot analysis using antibodies against GAPDH and LC3. Quantification of the relative intensity of LC3-II to GAPDH. Data from three independent experiments are represented as means ± SEM, ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. NT indicates no transfection. (C, D) HEK 293 cells were transfected with GFP, GFP-GR∗30 or GFP-PR∗30, along with mCherry-LC3 or mCherry-GABARAPL1. After 24 h, the cells were incubated with EBSS or normal culture medium for 1 h and visualized using fluorescence microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (E) Experimental design of assessing autophagy level in mouse model. (F) C57BL/6 mice at 4 months were injected in vivo -jetPEI mixed with or without GFP-PR∗30 in right lateral cerebral ventricle. Mice were sacrificed after 21 days and the brain lysates were subjected to immunoblot analysis using antibodies against GFP, Tubulin and LC3 ( n = 3 animals per group). (G) Quantification of the relative intensity of LC3-II to Tubulin in F. Data from three independent experiments are represented as means ± SD; ∗∗ P < 0.01, one-way ANOVA. (H) C57BL/6 mice at 2 months or 3 months were injected with or without AAV-GFP-GR∗30 in right lateral cerebral ventricle. Mice were sacrificed after 21 days and the brain lysates were subjected to immunoblot analysis using antibodies against GFP, Tubulin and LC3 ( n = 3 animals per group). (I) Quantification of the relative intensity of LC3-II to Tubulin in (H). Data from three independent experiments are represented as means ± SD; ∗∗ P < 0.01, one-way ANOVA. (J) C57BL/6 mice at 2 months were injected with BafiA1 (40 or 70 or 100 nmol). Mice were sacrificed after 24 h and subjected to immunostaining analysis using antibodies against LC3 ( n = 3 animals per group), yellow arrows indicate LC3-positive autophagosomes. (K) C57BL/6 mice at 2 months were injected with or without AAV-GFP-GR∗30 in right lateral cerebral ventricle and injected with 100 nmol BafiA1 after 20 days. Mice were sacrificed after 24 h and subjected to immunostaining analysis using antibodies against LC3 ( n = 3 animals per group), yellow arrows indicate LC3-positive autophagosomes. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 5 μm.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: C9orf72 -linked poly-GR and poly-PR inhibit the formation of autophagosomes under starvation condition. (A, B) HEK 293 cells were transfected with 0.1, 0.5 or 3.5 μg GFP-GR∗30, GFP-PR∗30 or GFP tag. After 48 h, the cells were incubated with Earle's balanced salt solution (EBSS) for 1 h, and cell lysates were subjected to immunoblot analysis using antibodies against GAPDH and LC3. Quantification of the relative intensity of LC3-II to GAPDH. Data from three independent experiments are represented as means ± SEM, ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. NT indicates no transfection. (C, D) HEK 293 cells were transfected with GFP, GFP-GR∗30 or GFP-PR∗30, along with mCherry-LC3 or mCherry-GABARAPL1. After 24 h, the cells were incubated with EBSS or normal culture medium for 1 h and visualized using fluorescence microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (E) Experimental design of assessing autophagy level in mouse model. (F) C57BL/6 mice at 4 months were injected in vivo -jetPEI mixed with or without GFP-PR∗30 in right lateral cerebral ventricle. Mice were sacrificed after 21 days and the brain lysates were subjected to immunoblot analysis using antibodies against GFP, Tubulin and LC3 ( n = 3 animals per group). (G) Quantification of the relative intensity of LC3-II to Tubulin in F. Data from three independent experiments are represented as means ± SD; ∗∗ P < 0.01, one-way ANOVA. (H) C57BL/6 mice at 2 months or 3 months were injected with or without AAV-GFP-GR∗30 in right lateral cerebral ventricle. Mice were sacrificed after 21 days and the brain lysates were subjected to immunoblot analysis using antibodies against GFP, Tubulin and LC3 ( n = 3 animals per group). (I) Quantification of the relative intensity of LC3-II to Tubulin in (H). Data from three independent experiments are represented as means ± SD; ∗∗ P < 0.01, one-way ANOVA. (J) C57BL/6 mice at 2 months were injected with BafiA1 (40 or 70 or 100 nmol). Mice were sacrificed after 24 h and subjected to immunostaining analysis using antibodies against LC3 ( n = 3 animals per group), yellow arrows indicate LC3-positive autophagosomes. (K) C57BL/6 mice at 2 months were injected with or without AAV-GFP-GR∗30 in right lateral cerebral ventricle and injected with 100 nmol BafiA1 after 20 days. Mice were sacrificed after 24 h and subjected to immunostaining analysis using antibodies against LC3 ( n = 3 animals per group), yellow arrows indicate LC3-positive autophagosomes. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 5 μm.

Article Snippet: GFP, GFP-TFEB, GFP-BCL2, GFP-LC3, GFP-Htt-60Q, GFP-Htt-150Q, GFP-BCL2, FLAG, FLAG-BECN1, mCherry, mCherry-GFP-LC3B, GFP-GAggagca (GA∗30), GFP-GRggaaga (GR∗30), GFP-PRccaaga (PR∗30), Flag-GAggagca (GA∗30), Flag-GRggaaga (GR∗30) and mCherry-PRccccgg (PR∗46) plasmids were described previously , , , , , , . mCherry-LC3B was a gift from Dr. David Rubinsztein (Addgene plasmid #40827). pMRX-IP-GFP-LC3-RFP-LC3ΔG was a gift from Dr. Mizushima (Addgene plasmid #84572).

Techniques: Transfection, Incubation, Western Blot, Fluorescence, Microscopy, Injection, In Vivo, Immunostaining

Poly-GR and poly-PR inhibit the autophagic clearance of poly-Q proteins. (A, B) HEK 293 cells were transfected with mCherry-GFP-LC3 and FLAG-GA∗30, FLAG-GR∗30 or FLAG tag. After 24 h, the cells were incubated with normal culture medium or EBSS for 1 h. Cells were visualized using confocal microscopy. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. The quantitative data of yellow dots (autophagosomes) or red dots (autolysosomes) are shown in (B). Data from three independent experiments represented as means ± SEM; ∗∗∗ P < 0.001, one-way ANOVA. (C) HEK 293 cells were transfected with GFP-Htt-60Q or GFP-Htt-150Q, along with FLAG-GA∗30, FLAG-GR∗30, mCherry-PR∗46 or FLAG tag (Mock). After 12 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then the cells were visualized using fluorescence microscopy. The quantitative data of poly-Q aggregates from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (D) Mouse embryonic fibroblast (MEF) cells or ATG5 KO MEF cells were similarly transfected with GFP-Htt-60Q, along with FLAG-GA∗30, FLAG-GR∗30, mCherry-PR∗46 or FLAG tag (Mock). Cells processed as in (C). The quantification data of poly-Q aggregates are shown as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOV. (E) HEK 293 cells were transfected with GFP-LC3-RFP-LC3ΔG and FLAG-GA∗30, FLAG-GR∗30, FLAG-PR∗30 or FLAG tag. After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. Statistical data from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: Poly-GR and poly-PR inhibit the autophagic clearance of poly-Q proteins. (A, B) HEK 293 cells were transfected with mCherry-GFP-LC3 and FLAG-GA∗30, FLAG-GR∗30 or FLAG tag. After 24 h, the cells were incubated with normal culture medium or EBSS for 1 h. Cells were visualized using confocal microscopy. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. The quantitative data of yellow dots (autophagosomes) or red dots (autolysosomes) are shown in (B). Data from three independent experiments represented as means ± SEM; ∗∗∗ P < 0.001, one-way ANOVA. (C) HEK 293 cells were transfected with GFP-Htt-60Q or GFP-Htt-150Q, along with FLAG-GA∗30, FLAG-GR∗30, mCherry-PR∗46 or FLAG tag (Mock). After 12 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then the cells were visualized using fluorescence microscopy. The quantitative data of poly-Q aggregates from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (D) Mouse embryonic fibroblast (MEF) cells or ATG5 KO MEF cells were similarly transfected with GFP-Htt-60Q, along with FLAG-GA∗30, FLAG-GR∗30, mCherry-PR∗46 or FLAG tag (Mock). Cells processed as in (C). The quantification data of poly-Q aggregates are shown as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOV. (E) HEK 293 cells were transfected with GFP-LC3-RFP-LC3ΔG and FLAG-GA∗30, FLAG-GR∗30, FLAG-PR∗30 or FLAG tag. After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. Statistical data from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA.

Techniques: Transfection, FLAG-tag, Incubation, Confocal Microscopy, Fluorescence, Microscopy

Poly-PR inhibits the initiation of autophagosomal biogenesis. (A) HEK 293 cells were transfected with ATG9-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were treated with EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. (B) HEK 293 cells were transfected with DFCP1-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were starved for 1 h with EBSS. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (C) The number of DFCP1 dots per cell in (B) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (D) HEK 293 cells were transfected with ATG16-mCherry and GFP-PR∗30 or GFP tag (Mock). After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (E) The number of ATG16 dots per cell was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (F) HEK 293 cells were transfected with ATG5-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h Then, the cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (G) The number of ATG5 dots per cell was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: Poly-PR inhibits the initiation of autophagosomal biogenesis. (A) HEK 293 cells were transfected with ATG9-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were treated with EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. (B) HEK 293 cells were transfected with DFCP1-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were starved for 1 h with EBSS. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (C) The number of DFCP1 dots per cell in (B) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (D) HEK 293 cells were transfected with ATG16-mCherry and GFP-PR∗30 or GFP tag (Mock). After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h. Then, cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (E) The number of ATG16 dots per cell was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA. (F) HEK 293 cells were transfected with ATG5-GFP and mCherry-PR∗46 or mCherry tag (Mock). After 48 h, the cells were incubated with normal culture medium or EBSS for 1 h Then, the cells were visualized using confocal microscopy. Scale bar, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (G) The number of ATG5 dots per cell was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01, one-way ANOVA.

Techniques: Transfection, Confocal Microscopy, Incubation

Poly-PR inhibits the autophagy by promoting the interaction between BECN1 and BCL2. (A, B) HEK 293 cells were transfected with GR∗30, PR∗46 or empty tag (Mock). After 48 h, cells lysates were subjected to immunoblot analysis using antibodies against BECN1 and GAPDH. The relative intensity of BECN1 to GAPDH are shown in (B). Data from three independent experiments are represented as means ± SEM; ns, not significantly different, one-way ANOVA. (C) HEK 293T cells were transfected with FLAG-BECN1 and GFP-BCL2 or GFP tag and mCherry-PR∗46 or mCherry tag. After 48 h, the supernatants of the cell lysates were used in immunoprecipitation assay using GFP antibody. Bound proteins were detected with GFP, FLAG and GAPDH antibodies. (D) HEK 293T cells were transfected with GFP-BCL2 and FLAG-BECN1 or FLAG tag and mCherry-PR∗46 or mCherry tag. After 48 h, the supernatants of the cell lysates were used in immunoprecipitation assay with FLAG antibody. Bound proteins were detected with GFP, FLAG and GAPDH antibodies. (E–G) HEK 293 cells were transfected with GFP-GR∗30, GFP-PR∗30 or GFP tag (Mock). After 48 h, cell lysates were subjected to immunoblot analysis using antibodies against phosphorylated BCL2 (S87) in (E) or phosphorylated BCL2 (S70) in (F), BCL2, LC3 and GAPDH. The relative intensity of phosphorylated BCL2 (S87) or (S70) to total BCL2 are shown in (G). Data from three independent experiments are represented as means ± SEM; ∗ P < 0.05; ∗∗ P < 0.01, one-way ANOVA. (H) HEK 293 cells were transfected with indicated siRNA for 48 h. Then the cells were re-transfected with mCherry-LC3 and GFP-PR∗30 or GFP tag. After 24 h, the cells were incubated with EBSS for 1 h. Then, cells were visualized using confocal microscope. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (I) The number of LC3 dots per cell in (H) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗∗ P < 0.001, one-way ANOVA. (J, K) HEK 293 cells were similarly transfected and treated as in (H). Then, cells lysates were subjected to immunoblot analysis using antibodies against BECN1, LC3 and GAPDH. The relative intensity of LC3-II to GAPDH are shown in (K). Data from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: Poly-PR inhibits the autophagy by promoting the interaction between BECN1 and BCL2. (A, B) HEK 293 cells were transfected with GR∗30, PR∗46 or empty tag (Mock). After 48 h, cells lysates were subjected to immunoblot analysis using antibodies against BECN1 and GAPDH. The relative intensity of BECN1 to GAPDH are shown in (B). Data from three independent experiments are represented as means ± SEM; ns, not significantly different, one-way ANOVA. (C) HEK 293T cells were transfected with FLAG-BECN1 and GFP-BCL2 or GFP tag and mCherry-PR∗46 or mCherry tag. After 48 h, the supernatants of the cell lysates were used in immunoprecipitation assay using GFP antibody. Bound proteins were detected with GFP, FLAG and GAPDH antibodies. (D) HEK 293T cells were transfected with GFP-BCL2 and FLAG-BECN1 or FLAG tag and mCherry-PR∗46 or mCherry tag. After 48 h, the supernatants of the cell lysates were used in immunoprecipitation assay with FLAG antibody. Bound proteins were detected with GFP, FLAG and GAPDH antibodies. (E–G) HEK 293 cells were transfected with GFP-GR∗30, GFP-PR∗30 or GFP tag (Mock). After 48 h, cell lysates were subjected to immunoblot analysis using antibodies against phosphorylated BCL2 (S87) in (E) or phosphorylated BCL2 (S70) in (F), BCL2, LC3 and GAPDH. The relative intensity of phosphorylated BCL2 (S87) or (S70) to total BCL2 are shown in (G). Data from three independent experiments are represented as means ± SEM; ∗ P < 0.05; ∗∗ P < 0.01, one-way ANOVA. (H) HEK 293 cells were transfected with indicated siRNA for 48 h. Then the cells were re-transfected with mCherry-LC3 and GFP-PR∗30 or GFP tag. After 24 h, the cells were incubated with EBSS for 1 h. Then, cells were visualized using confocal microscope. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (I) The number of LC3 dots per cell in (H) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗∗ P < 0.001, one-way ANOVA. (J, K) HEK 293 cells were similarly transfected and treated as in (H). Then, cells lysates were subjected to immunoblot analysis using antibodies against BECN1, LC3 and GAPDH. The relative intensity of LC3-II to GAPDH are shown in (K). Data from three independent experiments are represented as means ± SEM; ns, not significantly different; ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA.

Techniques: Transfection, Western Blot, Immunoprecipitation, FLAG-tag, Incubation, Microscopy

SW063058 alleviates C9orf72 arginine-containing DPR-induced autophagy inhibition through specifically targeting to BCL2–BECN1 protein complex. (A) HEK 293 cells were transfected with mCherry-LC3 and GFP or GFP-PR∗30, and then the cells were pre-treated with SW063058 at indicated concentrations for 11 h. The cells were incubated with EBSS for 1 h and visualized using fluorescence microscope. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (B) The number of LC3 dots per cell in (A) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗∗ P < 0.001, one-way ANOVA. (C) HEK 293 cells were similarly transfected and treated as (A). The cell lysates were subjected to immunoblot with indicated antibodies. (D) The relative intensities in C from three independent experiments were presented as means ± SD; ns, not significantly different; ∗ P < 0.05, one-way ANOVA. (E) HEK 293 cells were transfected with indicated plasmids and treated with 10 μmol/L SW063058 for 12 h. The supernatants of the cell lysates were used in immunoprecipitation assay using anti-FLAG antibody. ∗ indicates IgG heave chains. Right region: schematic model illustrating the disruption of the interaction between BECN1 and BCL2 by SW063058.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: SW063058 alleviates C9orf72 arginine-containing DPR-induced autophagy inhibition through specifically targeting to BCL2–BECN1 protein complex. (A) HEK 293 cells were transfected with mCherry-LC3 and GFP or GFP-PR∗30, and then the cells were pre-treated with SW063058 at indicated concentrations for 11 h. The cells were incubated with EBSS for 1 h and visualized using fluorescence microscope. Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (B) The number of LC3 dots per cell in (A) was counted, and 20–30 cells in each group from three independent experiments were statistically analyzed. Data are represented as means ± SEM; ns, not significantly different; ∗∗∗ P < 0.001, one-way ANOVA. (C) HEK 293 cells were similarly transfected and treated as (A). The cell lysates were subjected to immunoblot with indicated antibodies. (D) The relative intensities in C from three independent experiments were presented as means ± SD; ns, not significantly different; ∗ P < 0.05, one-way ANOVA. (E) HEK 293 cells were transfected with indicated plasmids and treated with 10 μmol/L SW063058 for 12 h. The supernatants of the cell lysates were used in immunoprecipitation assay using anti-FLAG antibody. ∗ indicates IgG heave chains. Right region: schematic model illustrating the disruption of the interaction between BECN1 and BCL2 by SW063058.

Techniques: Inhibition, Transfection, Incubation, Fluorescence, Microscopy, Western Blot, Immunoprecipitation, Disruption

C9orf72 arginine-containing DPRs impede neuronal autophagy. (A) Mouse cortical neurons (DIV 7) were transfected with mCherry-LC3 and GFP or GFP-PR∗30, and then were pre-treated with SW063058 for 4 h. The neurons were incubated with EBSS for 8 h prior to immunofluorescent assay with anti-MAP2 antibody (magenta) and Hoechst (cyan). Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (B) The number of LC3 dots in soma per neuron in (A) was counted, and 20 neurons in each group from three independent experiments were statistically analyzed. Data are represented as mean ± SEM; ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA. (C) Mouse cortical neurons (DIV 7) were transfected with GFP or GFP-PR∗30. Images of in situ PLA using rabbit anti-BECN1 and mouse anti-BCL2 antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bars, 10 μm. (D) The number of PLA dots per neuron in (C) was counted, and 20 somas of neuron in each group from three independent experiments were statistically analyzed. Data are represented as mean ± SEM; ∗∗∗ P < 0.001, one-way ANOVA. (E) The schematic model of this study. BECN1–BCL2 interaction regulates autophagy in cells. DPRs significantly inhibit neuronal autophagy by enhancing the interactions of BECN1 and BCL2. A small molecule compound SW063058 disrupts BECN1–BCL2 interaction, thereby restoring autophagy in DPR-expressing neurons.

Journal: Acta Pharmaceutica Sinica. B

Article Title: ALS-linked C9orf72 dipeptide repeats inhibit starvation-induced autophagy through modulating BCL2–BECN1 interaction

doi: 10.1016/j.apsb.2024.02.004

Figure Lengend Snippet: C9orf72 arginine-containing DPRs impede neuronal autophagy. (A) Mouse cortical neurons (DIV 7) were transfected with mCherry-LC3 and GFP or GFP-PR∗30, and then were pre-treated with SW063058 for 4 h. The neurons were incubated with EBSS for 8 h prior to immunofluorescent assay with anti-MAP2 antibody (magenta) and Hoechst (cyan). Scale bars, 10 μm. Insets were higher magnifications of the dashed box area. Scale bars, 2 μm. (B) The number of LC3 dots in soma per neuron in (A) was counted, and 20 neurons in each group from three independent experiments were statistically analyzed. Data are represented as mean ± SEM; ∗∗ P < 0.01; ∗∗∗ P < 0.001, one-way ANOVA. (C) Mouse cortical neurons (DIV 7) were transfected with GFP or GFP-PR∗30. Images of in situ PLA using rabbit anti-BECN1 and mouse anti-BCL2 antibodies. Blue: nuclei (DAPI); white dots: PLA positive puncta. Scale bars, 10 μm. (D) The number of PLA dots per neuron in (C) was counted, and 20 somas of neuron in each group from three independent experiments were statistically analyzed. Data are represented as mean ± SEM; ∗∗∗ P < 0.001, one-way ANOVA. (E) The schematic model of this study. BECN1–BCL2 interaction regulates autophagy in cells. DPRs significantly inhibit neuronal autophagy by enhancing the interactions of BECN1 and BCL2. A small molecule compound SW063058 disrupts BECN1–BCL2 interaction, thereby restoring autophagy in DPR-expressing neurons.

Techniques: Transfection, Incubation, In Situ, Expressing

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