Journal: Nature Communications

Article Title: Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability

doi: 10.1038/s41467-020-15156-5

Figure Lengend Snippet: In a through ( f ), endogenous Rap1 activity (Rap1A+B-GTP) was assessed through immunoprecipitation of GTP-bound Rap1 and immunoblotting against the indicated proteins. Cells had been subjected to 1 h depletion of all amino acids (−AA) ( a , e , f ), glucose ( b ) or serum ( c , e ), and re-stimulated with amino acids for 30 min ( a ), or treated for 1 h with 20 nM Rapamycin (Rapa), 50 nM Torin1 or DMSO control ( d ). Lysates were prepared from serum deprived HEK293T cells ( a ), HEK293A cells ( b – d ), RagA/B knockout HEK293A cells ( e ) or MEF cells deficient in GCN2 ( f ). Graphs represent relative immunoblot band intensity from n experiments: a , b , c , e , f n = 3, d n = 4. In a , d , e , f Rap1-GTP and total Rap1 were processed on separate blots due to technical reasons. Statistical data are presented as mean values ± s.e.m. n.s. = not significant ( P > 0.05); Student’s t -test; two-sided, unpaired. See Source Data File for statistics source data. Uncropped images of blots are shown in Supplementary Fig. .

Article Snippet: Torin1 (Tocris Bioscience, 4247) was used at a final concentration of 50 nM and Rapamycin (Calbiochem, 553210) at a final concentration of 20 nM.

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Control, Knock-Out

Journal: Nature Communications

Article Title: Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability

doi: 10.1038/s41467-020-15156-5

Figure Lengend Snippet: a U2OS cells were subjected to 50 nM Torin1 or amino acid starvation in the presence or absence of 100 μM CQ for 4 h, and separated into lysosome-enriched heavy membranes and cytosolic fractions, and analyzed by immunoblotting for the indicated proteins. Cells were pretreated with CQ for 1 h before treatment was initiated. Graph shows the quantification of lysosome fraction from three individual experiments. b , c Representative Z-stack projections and 3d-reconstructions of the change in lysosomal abundance in U2OS cells transfected with the indicated FLAG-Rap1 cDNAs after treatment with 20 μM CQ for 24 h. 3d-reconstruction was performed on all FLAG-Rap1 cDNA expressing cells in the field of view. The yellow arrow indicates one representative cell for which the 3d-reconstruction is shown below. The percentage change in lysosomal abundance is quantified in c . The number of cells analyzed to quantify lysosome abundance is shown in Supplementary Fig. . Scale bars: middle panel 10 μm, lower panel 20 μm. d , e Amino acid starvation induces F-actin rearrangements in a Rap1-dependent manner, as shown in Rap1-depleted U2OS cells stained with Phalloidin ( d ), counting at least 300 cells per condition across three individual experiments and scoring for cells with increased peripheral F-actin ( e ). Scale bar: 10 μm. In f , g lysosomal distribution was assessed in U2OS cells that had been amino acid starved for 3.5 h and treated with either DMSO or 0.1 μM latrunculin A (Lat A) for an additional 30 min (total 4 h of starvation). Quantifications are shown in g . Also see Supplementary movie . Scale bar: 10 μm. The microscopic fields imaged were randomly selected. In g n denotes the number of individual cells analyzed across three independent experiments and data are presented as mean values ± s.d. In a , c , e n denotes the number of individual experiments and data are presented as mean values ± s.e.m. n.s. = not significant ( P > 0.05); Student’s t -test; two-sided, unpaired ( a , c , e ), one-way ANOVA with Tukey’s post hoc test ( g ). See Source Data File for statistics source data. Uncropped images of blots are shown in Supplementary Fig. .

Article Snippet: Torin1 (Tocris Bioscience, 4247) was used at a final concentration of 50 nM and Rapamycin (Calbiochem, 553210) at a final concentration of 20 nM.

Techniques: Western Blot, Transfection, Expressing, Staining

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: Screening of autophagy inducers in neuronal PC12D cells using GFP-LC3-RFP autophagy flux probe. (A) Schematic illustration of establishment of PC12D cells stably expressing GFP-LC3-RFP autophagy flux probe. (B) PC12D cells expressing GFP-LC3-RFP were treated with 10 µM rapamycin (Rapa), 100 nM torin1, or 10 nM bafilomycin A 1 (Baf A 1 ) for 24 h. Scale bar: 20 µm. (C) PC12D cells expressing GFP-LC3-RFP were treated with the indicated concentrations of rapamycin, torin1, or bafilomycin A 1 for 24 h. The GFP:RFP fluorescence intensity ratio was measured using a high-content imager. (D) Schematic illustration of the chemical screen. (E) Scatter plot of the GFP:RFP ratio of each compound in the screen. (F) Bar graph showing the GFP:RFP ratios of the top 50 compounds. Green bars indicate compounds that significantly induced autophagy (p < 0.05, two-tailed Student’s t test compared to control). Data are shown as mean ± SD (n = 3). n.s., non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed one-way ANOVA with post hoc Dunnett’s test)

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Stable Transfection, Expressing, Fluorescence, Two Tailed Test, Control

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: Autophagy inducers used in this study

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Concentration Assay, Histone Deacetylase Assay, Glycoproteomics

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: SMK-17 induces autophagy in a MAP2 K inhibition- or MTOR-independent manner. (A) Chemical structure of SMK-17. (B, C) Western blotting analyses of NGF-differentiated PC12D cells treated with (B) 10 µM SMK-17 for the indicated times, (C) 10 µM SMK-17 in the presence or absence of 100 nM bafilomycin A 1 (Baf A 1 ) for 24 h with the indicated antibodies. (D) NGF-differentiated PC12D cells transfected with the mCherry-GFP-LC3 (tfLC3) plasmid vector were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 8 h. Autophagy flux was observed under confocal microscopy. Scale bar: 20 µm. (E, F) Western blotting analyses of NGF-differentiated PC12D cells treated with (E) 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 4 h, or (F) 100 nM torin1, 10 µM SMK-17, or 10 µM U0126 for 1 h with the indicated antibodies

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Inhibition, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: SMK-17 induces autophagy through TFEB activation. (A) Representative images and (B) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1, 10 µM SMK-17, 10 µM U0126, or 10 µM PD184352 for 1 h. ( C, D, E ) SMK-17 induces expression of TFEB target genes. (C) NGF-differentiated PC12D cells were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (D) Knockdown of TFEB in NGF-differentiated PC12D cells was confirmed with western blotting. (E) NGF-differentiated PC12D cells transfected with Tfeb siRNA or control siRNA were treated with 10 µM SMK-17 for 6 h followed by qRT-PCR analysis. (F) Involvement of TFEB in SMK-17-induced autophagy. NGF-differentiated PC12D cells expressing GFP-LC3-RFP were transfected with Tfeb siRNA or control siRNA and then treated with 10 µM SMK-17 for 24 h. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. Scale bar: 20 µm. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t test)

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Activation Assay, Nuclear Translocation Assay, Stable Transfection, Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Transfection, Control, Two Tailed Test

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: SMK-17 induces PRKC/PKC-dependent TFEB activation and clearance of intracellular aggregates. (A, B) Western blotting analyses of NGF-differentiated PC12D cells treated with (A) 10 µM SMK-17 or 100 nM phorbol 12-myristate 13-acetate (PMA) for the indicated times, or (B) 10 µM SMK-17 in the presence or absence of 5 µM PRKC inhibitor (PRKCi, Gö6983) for 3 h. Phosphorylation of PRKC substrates was detected by using p-(Ser) PRKC substrate antibody. (C) Representative images and (D) quantification of TFEB nuclear translocation assay results. NGF-differentiated PC12D cells stably expressing TFEB-GFP were treated with 100 nM torin1 or 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Scale bar: 20 µm. (E) Representative images and (F) quantification of LysoTracker Red DND-99 staining assay results. NGF-differentiated PC12D cells were treated with 100 nM torin1, 10 µM SMK-17 in the presence or absence of 5 µM PRKCi. Mean fluorescent intensity was quantified. Scale bar: 20 µm. (G) NGF-differentiated PC12D cells expressing GFP-LC3-RFP were treated with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Autophagy flux was evaluated by GFP:RFP ratio using a plate reader. (H) Representative images and (I) quantification of aggresome clearance assay results. NGF-differentiated PC12D cells were treated with MPP + for 16 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 8 h in the presence or absence of 5 µM PRKCi. The number of aggresoe dots per cell in each image was quantified. Scale bar: 20 µm. (J) Western blotting analysis of NGF-differentiated PC12D cells transiently transfected with GFP, GFP-HTTQ23, or GFP-HTTQ74 for 72 h. (K) Representative images and (L) quantification of mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with 100 nM torin1 or 10 µM SMK-17 for 24 h in the presence or absence of 5 µM PRKCi. Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. Data are shown as mean ± SD (n = 3). n.s., non-significant, *p < 0.05, ***p < 0.001 (two-tailed Student’s t test)

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Nuclear Translocation Assay, Stable Transfection, Expressing, Staining, Transfection, Mutagenesis, Two Tailed Test

Journal: Autophagy

Article Title: A chemical genomics-aggrephagy integrated method studying functional analysis of autophagy inducers

doi: 10.1080/15548627.2020.1794590

Figure Lengend Snippet: Activities of autophagy inducers in inhibition of aggresome formation and clearance of protein aggregates. (A) Representative images and (B) quantification of aggresome formation assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 24 h in the presence or absence of 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (C) Representative images and (D) quantification of aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with 10 µM SMK-17, 100 nM torin1, 10 µM rapamycin, 10 mM NAC, or 10 mM GSH for 8 h. The number of aggresome dots per cell in each image was quantified. Scale bar: 10 µm. (E) Quantification of the aggresome clearance assay results. RA-differentiated SH-SY5Y cells were treated with MPP + for 16 h prior to treatment with the indicated compounds for 8 h. See also Table 1 and Fig. S3A . The number of aggresome dots per cell in each image was quantified. (F) Quantification of the mutant HTT clearance assay results. NGF-differentiated PC12D cells were transiently transfected with GFP-HTTQ23 or GFP-HTTQ74 for 48 h prior to treatment with the indicated compounds for 24 h. See also Table 1 and Fig. S3B . Percentage of cells with GFP-HTT aggregates to GFP-positive cells was calculated in each sample. (G) Cytotoxicity of the autophagy inducers against primary cultured rat cortical neurons. Cells were treated with the indicated compounds for 24 h, and cytotoxicity was measured by LDH release assay. The data are expressed as a percentage of total amount of LDH analyzed in each plate. Data are shown as mean ± SD (n = 3 [ B, D, E, F ], n = 5 [ G ]). ### p < 0.001 (two-tailed Student’s t test compared to untreated control [Ctrl]). n.s., non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t test [ B, D, E, F ], one-way ANOVA with post hoc Dunnett’s test [ G ])

Article Snippet: MG132 (10012628) and torin1 (10997) were purchased from Cayman Chemical.

Techniques: Inhibition, Tube Formation Assay, Mutagenesis, Transfection, Cell Culture, Lactate Dehydrogenase Assay, Two Tailed Test, Control