lentiviral constructs  (Addgene inc)

Journal: Theranostics

Article Title: Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models

doi: 10.7150/thno.88718

Figure Lengend Snippet: ALT001 induces mitophagy through the ULK1-Rab9 alternative mitophagy pathway. (A) BEAS-2B cells were treated with ALT001 (15 μM) for 12 h, and western blot analysis was performed using the indicated antibodies. Quantitative analysis of the protein levels shown on the right as the mean ± SD. (B) BEAS-2B cells expressing YFP-Rab9 were treated with ALT001 (15 μM) for 12 h. YFP-Rab9 puncta were analyzed by confocal microscopy, and cells with more than forty YFP-Rab9 puncta were considered positive cells. The percentage of Rab9 puncta-positive cells from three experiments is shown on the right as the mean ± SD (vehicle (Veh), n = 54; ALT001, n = 45). Scale bar: 10 μm. (C) BEAS-2B cells expressing YFP-Rab9 were treated with ALT001 (15 μM) for 12 h and stained with MitoTracker Red. Fluorescence images were analyzed by confocal microscopy. Arrows indicate YFP-Rab9-containing mitochondria. Scale bar: 10 μm. The boxed regions are enlarged on the right. Scale bars: 5 μm (right enlarged). The number of YFP-Rab9 puncta-containing mitochondria per cell from three experiments is shown on the right as the mean ± SD (vehicle (Veh), n = 44; ALT001, n = 27). (D-E) BEAS-2B cells expressing mt-Keima (D) and BEAS-2B cells (E) were treated with ALT001 (15 μM) together with brefeldin A (BFA; 1 μg/ml) for 12 h, and mitophagy levels were analyzed by flow cytometry (D) . Western blot analysis was performed using the indicated antibodies (E) . Quantitative analysis of the protein levels shown on the right as the mean ± SD. (F-G) BEAS2B cells expressing shULK1 (F) or shRab9 (G) were treated with ALT001 (15 μM) for 12 h, and mitophagy levels were analyzed by flow cytometry. The results from three biological replicates (A, B, F, G) or four biological replicates (D) are shown as the mean ± SD. Significance was determined by Student's t test (A, B, C) or two-way ANOVA (D, E, F, G) with Šidák's multiple-comparison test. ** P < 0.01; *** P < 0.001. **** P < 0.0001. Numbers below the corresponding blot represent densitometric analysis normalized to Actin. Blots are representative of three to four biological replicates.

Article Snippet: For ULK1 knockdown, lentiviral constructs containing ULK1 shRNA (#27633) were obtained from Addgene.

Techniques: Western Blot, Expressing, Confocal Microscopy, Staining, Fluorescence, Flow Cytometry, Comparison

Journal: Theranostics

Article Title: Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models

doi: 10.7150/thno.88718

Figure Lengend Snippet: Specific induction of mitophagy by ALT001 independent of the canonical mitophagy pathway. (A-B) BEAS-2B cells expressing mt-Keima (A) or Keima (B) were treated with ALT001 (15 μM) for 18 h, and mitophagy levels were analyzed by confocal microscopy. Scale bar: 10 μm. (C) HeLa cells expressing Parkin and mitoYFP, ER-GFP, Golgi-eGFP, or Turquoise2-Peroxi were treated with ALT001 (15 μM) for 24 h. Scale bar: 10 μm. (D) BEAS-2B cells were treated with ALT001 (15 μM) for 24 h, and western blot analysis was performed using the indicated antibodies. Numbers below the corresponding blot represent densitometric analysis normalized to Actin. (E-F) BEAS-2B cells were treated with ALT001 (15 μM) for 24 h and further incubated until the indicated time points. Mitochondrial mass was measured by NAO staining (E) , and western blot analysis was performed using the indicated antibodies (F) . Numbers below the corresponding blot represent densitometric analysis normalized to Actin. (G) BEAS-2B cells were treated with either CCCP (10 μM) or ALT001 (15 μM) for the indicated time and western blot analysis was performed using LC3B and Actin antibodies. (H) HEK293 cells expressing EGFP-LC3 were treated with either CCCP (10 μM) or ALT001 (15 μM) for the indicated time, and EGFP-LC3 puncta were analyzed by confocal microscopy. The LC3 puncta number per cell from three experiments is shown as the mean ± SD (0 h, n = 40; ALT001 6 h, n = 29; 24 h, n = 31; CCCP 6 h, n = 39; CCCP 24 h, n = 21). (I) Wild-type (WT) and ATG7 knockout (ATG7 KO) HeLa cells expressing Parkin were treated with either CCCP (10 μM) for 2 h or ALT001 (15 μM) for 24 h, and mitophagy levels were analyzed by flow cytometry. (J) BEAS-2B expressing mt-Keima cells expressing control nontargeting shRNA (shNT) or PINK1 shRNA (shPINK1) were treated with either CCCP (10 μM) for 6 h or ALT001 (15 μM) for 24 h, and mitophagy levels were analyzed by flow cytometry. The results from three biological replicates (A-F, I-J) or four biological replicates (G) are shown as the mean ± SD. Significance was determined by Student's t test (A-C) or one-way (E, H) two-way ANOVA (I, J) with Šidák's multiple-comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. NS, not significant.

Article Snippet: For ULK1 knockdown, lentiviral constructs containing ULK1 shRNA (#27633) were obtained from Addgene.

Techniques: Expressing, Confocal Microscopy, Western Blot, Incubation, Staining, Knock-Out, Flow Cytometry, Control, shRNA, Comparison

Journal: Theranostics

Article Title: Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models

doi: 10.7150/thno.88718

Figure Lengend Snippet: ALT001 induces mitophagy through the ULK1-Rab9 alternative mitophagy pathway. (A) BEAS-2B cells were treated with ALT001 (15 μM) for 12 h, and western blot analysis was performed using the indicated antibodies. Quantitative analysis of the protein levels shown on the right as the mean ± SD. (B) BEAS-2B cells expressing YFP-Rab9 were treated with ALT001 (15 μM) for 12 h. YFP-Rab9 puncta were analyzed by confocal microscopy, and cells with more than forty YFP-Rab9 puncta were considered positive cells. The percentage of Rab9 puncta-positive cells from three experiments is shown on the right as the mean ± SD (vehicle (Veh), n = 54; ALT001, n = 45). Scale bar: 10 μm. (C) BEAS-2B cells expressing YFP-Rab9 were treated with ALT001 (15 μM) for 12 h and stained with MitoTracker Red. Fluorescence images were analyzed by confocal microscopy. Arrows indicate YFP-Rab9-containing mitochondria. Scale bar: 10 μm. The boxed regions are enlarged on the right. Scale bars: 5 μm (right enlarged). The number of YFP-Rab9 puncta-containing mitochondria per cell from three experiments is shown on the right as the mean ± SD (vehicle (Veh), n = 44; ALT001, n = 27). (D-E) BEAS-2B cells expressing mt-Keima (D) and BEAS-2B cells (E) were treated with ALT001 (15 μM) together with brefeldin A (BFA; 1 μg/ml) for 12 h, and mitophagy levels were analyzed by flow cytometry (D) . Western blot analysis was performed using the indicated antibodies (E) . Quantitative analysis of the protein levels shown on the right as the mean ± SD. (F-G) BEAS2B cells expressing shULK1 (F) or shRab9 (G) were treated with ALT001 (15 μM) for 12 h, and mitophagy levels were analyzed by flow cytometry. The results from three biological replicates (A, B, F, G) or four biological replicates (D) are shown as the mean ± SD. Significance was determined by Student's t test (A, B, C) or two-way ANOVA (D, E, F, G) with Šidák's multiple-comparison test. ** P < 0.01; *** P < 0.001. **** P < 0.0001. Numbers below the corresponding blot represent densitometric analysis normalized to Actin. Blots are representative of three to four biological replicates.

Article Snippet: For ULK1 knockdown, lentiviral constructs containing ULK1 shRNA (#27633) were obtained from Addgene.

Techniques: Western Blot, Expressing, Confocal Microscopy, Staining, Fluorescence, Flow Cytometry, Comparison

Journal: Theranostics

Article Title: Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models

doi: 10.7150/thno.88718

Figure Lengend Snippet: ALT001 restores mitochondrial function in the APP Swe/Ind mutant cellular model. (A-G) SH-SY5Y cells expressing the APP Swe/Ind mutant or control plasmid were treated with ALT001 (15 μM) for 12 h. After 48 h of recovery, mitochondrial superoxide levels were measured using MitoSOX Red staining (A) , and ATP levels were assessed (B) . Cells were analyzed by transmission electron microscopy (C) . Arrows indicate damaged mitochondria. Scale bars: 5 μm (upper). The boxed regions are enlarged in the bottom panel. Scale bars: 1 μm (bottom). The proportion of damaged mitochondria per cell is shown on the right as the mean ± SD (control plasmid, vehicle, n = 18; APP Swe/Ind, vehicle, n = 21; APP Swe/Ind, ALT001, n = 19). Representative mitochondrial respiration, analyzed by an XF-24 analyzer with five samples per group, is shown (D) . Basal respiration (E) , ATP production (F) , and maximal respiration (G) from mitochondrial respiration analyses are shown as the mean ± SD. (H-I) SH-SY5Y cells expressing the APP Swe/Ind mutant or control plasmid (-) were treated with ALT001 (15 μM) alone or cotreated with bafilomycin A1 (BafA1; 100 nM) for 12 h. The mitochondrial superoxide levels were measured using MitoSOX Red staining (H) , and ATP levels (I) were determined after 48 h of recovery. (J-K) SH-SY5Y cells stably expressing shRab9 or control shRNA (shNT) were transfected with APP Swe/Ind mutant or control plasmid and treated with ALT001 (15 μM) for 12 h. After 48 h of recovery, mitochondrial superoxide levels were measured using MitoSOX Red staining (J) , and ATP levels were assessed (K) . The results from four biological replicates (A, B, H, I) or five biological replicates (D-G, J, K) are shown as the mean ± SD. Significance was determined by one-way (A-C, E-G) or two-way ANOVA ( H-K ) with Šidák's multiple-comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; NS, not significant.

Article Snippet: For ULK1 knockdown, lentiviral constructs containing ULK1 shRNA (#27633) were obtained from Addgene.

Techniques: Mutagenesis, Expressing, Control, Plasmid Preparation, Staining, Transmission Assay, Electron Microscopy, Stable Transfection, shRNA, Transfection, Comparison

Journal: Theranostics

Article Title: Selective induction of Rab9-dependent alternative mitophagy using a synthetic derivative of isoquinoline alleviates mitochondrial dysfunction and cognitive deficits in Alzheimer's disease models

doi: 10.7150/thno.88718

Figure Lengend Snippet: AAV shRNA-mediated knockdown of Rab9 abolishes ALT001-mediated LTP restoration in 5×FAD mice. (A) FVB-mtKeima mice were injected with Rab9 shRNA AAV (AAV shRab9) or control shRNA AAV (AAV shNT) into the hippocampus. After 2 weeks, mice were treated with ALT001 (1 mg/kg) or vehicle (Veh) via intranasal administration daily for 7 days, and mt-Keima fluorescence was analyzed by confocal microscopy (AAV shNT vehicle, n = 4; AAV shNT ALT001, n = 4; AAV shRab9 shNT, n = 4, AAV shRab9 ALT001 n = 7). Scale bar: 50 μm. Quantified mitophagy levels from mice are shown on the right as the mean ± SD. ( B ) Four-month-old 5×FAD male mice were injected with Rab9 shRNA AAV (shRab9) or control shRNA AAV (shNT) into the hippocampus. After 2 weeks, the mice were treated with ALT001 (1 mg/kg) via intranasal administration daily for 4 weeks, and LTP analyses were performed (shRab9; red circle, n = 6, shNT; black circle, n = 6). Error bars indicate the SEM. ( C ) Schematic model for ALT001-mediated amelioration of mitochondrial dysfunction in Alzheimer's disease through alternative mitophagy. Significance was determined by one-way ANOVA with Šidák's multiple-comparison test. * P < 0.05.

Article Snippet: For ULK1 knockdown, lentiviral constructs containing ULK1 shRNA (#27633) were obtained from Addgene.

Techniques: shRNA, Knockdown, Injection, Control, Fluorescence, Confocal Microscopy, Comparison

Journal: PLoS ONE

Article Title: Synergistic Antitumor Effects of Endostar in Combination with Oxaliplatin via Inhibition of HIF and CXCR4 in the Colorectal Cell Line SW1116

doi: 10.1371/journal.pone.0047161

Figure Lengend Snippet: SW1116 cells were transduced with pLKO.1-shRNA-CXCR4. Control cells were transduced with vector pLKO.1. ( A–B ) Western blots of whole cell lysates for CXCR4 expression of SW1116 after CXCR4 knockdown. *** p <0.001 versus control. Proliferation ( C ), Matrigel adhesion ( D ) and invasion assay ( E ) were performed, showing CXCR4 contributes to proliferation, adhesion and invasion in SW1116 cells. ** p <0.01/*** p <0.001 versus control (n = 3).

Article Snippet: For production of recombinant lentivirus, 1×10 6 HEK 293T cells were co-transfected with 6 µg of target vector pLKO.1-shRNA, 4 µg of psPAX2 (12260; Addgene) and 2 µg pMD2G-VSVG (12259; Addgene) and incubated for 48 hours.

Techniques: Transduction, shRNA, Plasmid Preparation, Western Blot, Expressing, Invasion Assay