EMBO/soda-nel huggingface dataset:
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 2

Figure 2. Cand2 is translationally upregulated during cardiac pathological stress.

(A) Venn diagram showing a group of transcripts translationally upregulated in vivo 2 days after TAC (DEGs - Differentially Expressed Genes) and regulated by mTORC1 in vitro in NRCMs treated with Torin 1. Table with top translationally regulated mTOR-dependent transcripts in vitro after Torin 1 treatment of NRCMs and in vivo after TAC. Cand2 highlighted in red.

(B) Scatter plot of Ribo-seq vs. RNA-seq upon NRCMs treatment of Torin 1 shows Cand2 among translationally downregulated mRNAs. FC - fold change, Veh - vehicle.

(C) Protein expression of Cand2 in NRCMs treated with PE for 3 and 24 h, and 24h of Torin 1 treatment. mTORC1 induction was monitored by downstream targets, p4EBP1 and pS6K proteins. rpS5 - example of mTOR-dependent mRNA used as a positive control. Csq - calsequestrin used as a houskeeping protein.

(D) Cand2 mRNA levels in NRCMs treated with PE and Torin 1 measured by RT-qPCR. n=6-9 of biological replicates per conditions.

(E) Scatter plot of Ribo-seq vs. RNA-seq 2 days after TAC showing an increase of Cand2 translation
(F, G) Ribo-seq and RNA-seq expression data of Cand2 in sham and TAC operated animals (CPM - count per million). One-way ANOVA, ** - P ≤ 0.01, n=2-4 biological replicates per group.

(H) Immunoblots of Cand2 expression in left ventricles 2 days post-TAC and after injection with Torin 1. S235/236rpS6 was used as a positive control of mTOR-dependent mRNA. Csq - calsequestrin used as a houskeeping protein.

(I) Cand2 mRNA levels 2 days after TAC and after Torin1 injection measured by RT-qPCR. n=4-6 biological replicates per mice group.

(J) Schematic representation of reporter consisting of human Cand2 5'UTR and downstream Renilla luciferase coding sequence. A potential TOP-like motif is highlighted in a grey box. Black arrows indicate transcription start sites in the adult heart according to the Database of Transcriptional Start Sites (Release 10.1). The effect of Cand2 5'UTR on translation measured by luciferase activity in normal conditions and after mTORC1 block with Torin 1. eEF2 - positive control of reporter with defined 5'TOP motif and non-TOP 5'UTR of β-actin used as a negative control. Total protein content in lysates used for luciferase assay. t-test, n=4-5 biological replicates per group, ** - P ≤ 0.01, *** - P ≤ 0.001.

Data information: Error bars indicate means ± SEM
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 3

Figure 3. Cand2 is a muscle-specific protein

(A) Representative immunoblot of Cand2 protein levels in mouse organs.

(B) Cand2 expression in mouse organs measured by RT-qPCR. n=3 biological replicates.

(C) Immunofluorescent staining of paraffin sections from hearts from WT and Cand2 KO mice. CAND2 (stained in red), ACTIN (stained in green) and nuclei (stained with DAPI, blue) in. Scale bar 20μm

(D) Representative immunoblot of Cand2 protein from different cardiac cell types CM- cardiomyocytes, EC - endothelial cells, FB - fibroblast.

(E) Immunofluorescence of adult rat cardiomyocytes of Cand2 (stained in red), sarcomeric actin (stained in green), and nuclei (stained with DAPI blue). Scale bar 50μm.

(F) Immunoblot of subcellular fractionation of left ventricle (LV). Gapdh and Histone 3D (H3D) used as cytoplasmic and nuclear markers, respectively. WCL - whole cell lysate, cyt - cytoplasmic fraction, nuc - nuclear fraction.
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 4

Figure 4. Cand2 is required for cardiac hypertrophy induction.

(A) Representative immunoblot of Cand2 KD in NRCMs with Cand2-targeting siRNA (siScr - control scrambled siRNA).

(B) Cand2 OE in NRCMs (Cand2 AAV6; Ctr AAV - control AAV6 vector) analyzed by Western blot.

(C) Cell surface area measurement of NRCM after Cand2 KD and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments.Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile.

(D) RT-qPCR analysis of Nppa transcript level after Cand2 KD and PE treatment of NRCMs. Analysed by One-way ANOVA. n=11-12 biological replicates per conditions.

(E) NRCMs size measurement after Cand2 OE and 24h PE stimulation. Analyzed by One-way ANOVA. n>150 cells from n=3 independent experiments. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile.

(F) Nppa mRNA levels in vitro after Cand2 OE and PE treatment measured by RT-qPCR. Analysed by One-way ANOVA. n=5-9 biological replicates per conditions.

(G) Ejection fraction 2 weeks after TAC in WT and Cand2 KO mice measured by echocardiography. Analyzed by One-way ANOVA. n=5-13 biological replicates per group.

(H) Fractional shortening 2 weeks after TAC in WT and Cand2 KO mice measured by echocardiography. Analyzed by One-way ANOVA. n=5-13 biological replicates per mice group.

(I) LV weight to body weight ratio of WT and Cand2 KO mice subjected to sham and TAC surgeries. Analyzed by One-way ANOVA. n=7-11 biological replicates per condition.

(J) Nppa and Nppb mRNA levels 4 weeks after TAC in WT and Cand2 KO mice measured by RT-qPCR. Analyzed by One-way ANOVA. n=6-13 mice per group.

Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 5

Figure 5. Cand2-dependent Grk5 expression.

(A) Volcano plot of up - (log2-fold change for transcripts with counts per million, CPM >1; red dots) and downregulated (log2-fold change CPM <1; blue dots) transcripts in LV after Cand2 KO. Differential expression analysis performed with edgeR; Benjamini-Hochberg method for p-value calculation, cut off 0.05. Cand2 and Grk5 marked among depleted transcripts in Cand2 KO vs. WT mice.

Enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG Pathways) terms for differentially expressed genes in LV of Cand2 KO vs. WT mice.

(B) Immunoblots and quantification of Grk5 protein levels in vitro after Cand2 KD with siRNA and after Cand2 OE with AAV6. t-test, n=4-5 biological replicates per group.

(C) Grk5 mRNA levels in NRCMs depleted of and overexpressing Cand2 measured by RT-qPCR. t-test, n=6-9 biological replicates.

(D) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 4 weeks after sham and TAC analyzed by immunoblotting.

(E) Quantification of Grk5 protein levels and Grk5 mRNA levels in indicated conditions. Analyzed by One-way ANOVA, n=4-15 biological replicates .

(F) Representative immunoblot of Grk5 protein levels and quantification in cytoplasmic and nuclear fractions from left ventricle lysates of WT and Cand2 KO mice after TAC surgery. Lamin B - nuclear marker, Gapdh - cytoplasmic marker. Analyzed by One-way ANOVA, n=3 biological replicates.

Data information: Error bars indicate means ± SEM. * - P ≤ 0.05, *** - P ≤ 0.001.
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 6

Figure 6. Cand2 regulates MEF2 transcriptional activity.

(A) Luciferase-based quantification of MEF2 activity after Cand2 KD in NRCMs. Analysed by t-test, n=3 biological replicates.

(B) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs with Cand2 KD for indicated groups. PE treatment for 24h (50uM). Analyzed by One-way ANOVA, n=6 biological replicates.

(C) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs after Cand2 OE. Analyzed by t-test, n=7-8 biological replicates per conditions.

(D) RT-qPCR analysis of Xirp2 mRNA levels in LVs of WT and Cand2 KO mice after sham and TAC surgeries. Analyzed by One-way ANOVA, n=6-10 biological replicates per mice group.

(E) Grk5 protein and mRNA levels in NRCMs as well as MEF2-luciferase activity after 24h of Torin 1 treatment. Analyzed by t-test, n=4-6 biological replicates per conditions. Torin1 activity measured by protein levels of phosphorylated ribosomal S6 protein (S235/236rpS6).

(F) Cand2 and Grk5 protein levels after TSC2 KD Analyzed by Western blot as well as MEF2-luciferase activity after TSC2 knockdown. Analyzed by t-test, n=5-6 biological replicates per conditions.

(G) Cand2 and Grk5 protein levels and Raptor transcript level in NRCMs after Raptor KD analyzed by Western blot. S235/236rpS6 - phosphorylated ribosomal S6 protein, downstream target of mTORC1. Analyzed by t-test, n=3 biological replicates

(H) Quantifications of Cand2 and Grk5 protein levels level in NRCMs depleted of Raptor. Analyzed by t-test, n=3 biological replicates per conditions.

(I) Grk5, Nppa, and MEF2 targets Nr4a1 and Xirp2 mRNA levels in NRCMs after Raptor KD. Analyzed by t-test, n=3 biological replicates per conditions.

(J) Raptor and Grk5 protein levels after Raptor KD and adenoviral Grk5 overexpression.

(K) Cell surface area measurement of NRCM for indicated groups. Analyzed by One-way ANOVA. n=3 independent experiments, n>150 cells.

(L) RT-qPCR analysis of MEF2 targets in NRCMs after Raptor KD and Grk5 overexpression. Analyzed by One-way ANOVA. n=6-8 biological replicates per conditions.

Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 7

Figure 7. Cand2 and Cullin1 neddylation modulate Grk5 protein level.

(A) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in NRCMs after Cand2 overexpression. Analysed by t-test, n=3 biological replicates per conditions.

(B) Cul1 neddylation analyzed by Western blot in NRCMs depleted of Cand2. NRCMs were treated with MLN4924 (100 nM) to block Cul1 neddylation. Quantification of neddylated and unnedylated Cul1 protein levels after MLN4924 treatment for 24h. t-test, n=4 biological replicates.

(C) Cell size area measurement of NRCMs after Cand2 KD and neddylation inhibition (MLN). Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. Violin plot: the middle horizontal lane - median, upper lane - 3rd quartile, lower lane - 1st quartile.

(D) Grk5 expression in NRCMs upon Cand2 KD and Cul1 neddylation inhibition (MLN) analysed by immunoblot and RT-qPCR. Analyzed by One-way ANOVA, n=4-5 biological replicates.

(E) The half-life of Grk5 in HeLa cells overexpressing Cand2 measured by cycloheximide chase. n=2 biological replicates.

(F) Representative immunoblot and quantification of Grk5 protein levels as well as quantification of mRNA levels after Cul1 KD. t-test, n=4 biological replicates.

(G). Immunoblot after Cand2 KD and Grk5 OE. Cell surface area measurement of NRCM for indicated groups. Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. The middle horizontal lane on violin plot - median, upper lane - 3rd quartile, lower - 1st quartile.

(H) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA, n= 6 biological replicates.

(I) Immunoblot after Cand2 OE and Grk5 knockdown. Cell surface area measurement of NRCM for indicated groups. Analyzed by One-way ANOVA. n=3 independent experiments with >150 cells. The middle horizontal lane on violin plot - median, upper lane - 3rd quartile, lower - 1st quartile.

(J) mRNA levels of MEF2-dependent genes Nr4a1 and Xirp2 in NRCMs for indicated groups. Analyzed by One-way ANOVA. n=6 biological replicates.

Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling (2021)
Figure 8

Figure 8. Cand2 regulates Grk5 levels in vivo after TAC.

(A) Quantification of indicated mRNA levels 2 days after TAC measured by RT-qPCR. Analyzed by One-way ANOVA. n=5-7 biological replicates per group.

(B) LV weight to body weight ratio (mg/g) of WT and Cand2 KO mice subjected to sham and TAC surgeries after 2 days. Analyzed by One-way ANOVA. n=5-7 biological replicates per group.

(C) Grk5 protein levels in vivo in LVs of WT and Cand2 KO mice 2 days after sham and TAC analyzed by immunoblotting as well as quantification of mRNA levels analyzed by RT-qPCR. Analyzed by One-way ANOVA. n=3-5 biological replicates per group.

(D) Representative immunoblot and its quantification of protein levels of neddylated and unnedylated Cul1 in Cand2 KO mice compared to WT mice. Analyzed by t-test. n=5-8 mice per group. Cul1 neddylation was inhibited in C2C12 by MLN4924 (-/+ MLN) for neddylated Cul1 band detection.

(E) Model of Cand2-dependent stabilization of Grk5 by Cul1 neddylation inhibition in mTOR-dependent manner.

Data information: Error bars indicate means ± SEM; * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001.

Expanded View Figure Legends
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 2

Figure 2: ATG9A interacts with an ULK1-independent ATG13 complex that includes ATG101. (A) Schematic representation of ATG13 mutations used in the study. (B) HA-ATG9A-BirA* was expressed in HCT-116 ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with WT ATG13 or ATG13 ∆HORMA. Cells were grown in full DMEM media, treated with 50 µM biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests. (C) Cells were treated as in panel B but included reconstitution with ATG13 ∆2AA mutant. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests. (right). (D) HA-ATG9A-BirA* was overexpressed in WT and ULK1/2 Double KO MEFs. Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies. The graph on right shows quantification of normalized ATG13 infrared signal. Mean ± SEM. n=3 (biological replicates). Significance measured using one-sample t-test compared to hypothetical mean of 1 (right). ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001.
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 3

Figure 3: The loss of ATG13 causes a shift in colocalization of ATG9A with organelle and autophagy markers and triggers an accumulation of ATG9A with p62/SQSTM1. (A)-(G) Representative images of ATG9A colocalization with different organelle and autophagy markers. HCT-116 ATG9A-HA KI-ATG13 WT or ATG13 KO cells were grown in full DMEM media, fixed and labeled with antibodies for HA and indicated organelle/cellular markers and imaged (Scaler bar=10 µm). (H) Quantification of ATG9A colocalization with indicated organelle markers of golgi (GOLGIN97), mitochondria (TUFM), ER (PDIA3), endosomal system (VPS26A), early endosome (EEA1), lysosome (LAMP1), autophagy adaptor (p62), and autophagosome markers (ATG16L1, WIPI4, ATG2A, LC3II, WIPI2) respectively. Mean ± SEM, n=1 independent experiment with 30 technical replicates. Images for p62 colocalization (G) are from the same single independent experiment as Figures 4C and 5C.
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 4

Figure 4: The loss of ATG13 and ATG101 result in an accumulation of ATG9A at large clusters of p62/SQSTM1. (A) Endogenous p62/SQSTM1 level in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated antibodies. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting (left). The graph on right shows quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) Endogenous p62/SQSTM1 level in HEK-293T ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO clones was measured by immunoblotting with indicated proteins. Cells were grown in full DMEM media, treated with or without 100 nM Bafilomycin for 24 hrs and whole cell lysates were subjected to immunoblotting. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (C) Confocal images of ATG9A colocalization with p62/SQSTM1. HCT-116 ATG9-HA KI-ATG13 WT, ATG13 KO, ATG101 KO, ATG9A KO and FIP200 KO cells were grown in full DMEM media, fixed, labeled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar 10 µm). ATG101 KO was stained and quantified separately. Images are from the same single independent experiment as Figures 3G and 5C. (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1. Mean ± SEM, n=1 independent experiment with 30 technical replicates. ATG101 KO was stained and quantified separately. Mean ± SEM, n=1 independent experiment with 30 technical replicates.
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 5

Figure 5: ATG13-mediated rescue of ATG9A accumulation at p62/SQSTM1 clusters requires the ATG13 HORMA domain but is independent of ULK1. (A) Endogenous p62/SQSTM1 level was measured in HCT-116 ATG9A-HA KI-ATG13 WT, ATG13 KO OR ATG13 KO cells reconstituted with ATG13 WT, ATG13 ΔHORMA and ATG13 Δ2AA by immunoblotting with indicated proteins (left). Cells were grown in full DMEM media and whole cell lysates were subjected to immunoblotting (left). Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests (right). (B) HCT-116 cells in A were incubated in EBSS for 4 hours then processed and analyzed as in A. (C) Confocal images of ATG9A colocalization with p62/SQSTM1 in HCT-116 ATG9A-HA KI- ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and p62/SQSTM1 and imaged (Scale bar=10 µm). Images are from the same single independent experiment as Figures 3G and 4C. (D) Quantification of average surface area of p62/SQSTM1 puncta in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates. A break was inserted in the Y axis to accommodate the wide range of p62/SQSTM1 puncta sizes. (E) Quantification of ATG9A colocalization with p62/SQSTM1 in C. Mean ± SEM, n=1 independent experiment with 30 technical replicates.
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 6

Figure 6: Loss of ATG13 results in increased ATG9A recruitment to stalled clusters of p62/SQSTM1. (A) Live cell imaging of HCT116 ATG13 WT and HCT116 ATG13 KO cells stably expressing mRuby2-ATG9A and EGFP-p62/SQSTM1. Representative montages show mRuby2-ATG9A recruitment to EGFP-p62/SQSTM1 puncta (Scale bar=5 µm). (B) Confocal images of Ubiquitin colocalization with p62/SQSTM1 in HCT116 ATG13 WT and HCT116 ATG13 KO cells. Cells were grown in full DMEM media, fixed, labelled with antibodies for Ubiquitin and p62/SQSTM1 and imaged (Scale bar=10 µm) (left). Quantification of Ubiquitin colocalization with p62/SQSTM1 (right). Mean ± SEM, n=1 independent experiment with 30 technical replicates. (C) Modified pulse chase experiment. HEK293T ATG13 WT and HEK293T ATG13 KO cells stably expressing HA ATG9A-BirA* were pulse labeled with biotin for 24 hours, media was replaced with full DMEM and streptavidin pulldown was performed at indicated time points. Quantification of normalized p62 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using Student's t test (bottom). ns=p>0.05, *=p≤0.05, **=p≤0.01, ***=p≤0.001, ****= p≤0.0001.
BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1 clusters (2021)
Figure 7

Figure 7: A split-mVenus approach captures an ATG13-ATG101 interaction with ATG9A. (A) Schematic representation of bimolecular fluorescence complementation (BiFC) assay by using split-mVenus approach. (B) HCT-116 ATG9A-HA KI cells with overexpressed GFP only, VenusC-ATG101-3X FLAG only, VenusN-ATG13-Myc only, VenusN-ATG13 Δ2AA only or both Venus N and C halves together were subjected to immunoprecipitation with GFP-trap nano body resin. Immunoblotted for indicated proteins. (C) Confocal images of Venus colocalization with ATG9A and ULK1 in HCT116 ATG9A-HA KI ATG13-ATG101 double KO cells stably expressing VenusC-ATG101-3X FLAG and VenusN-ATG13-Myc or VenusN-ATG13 Δ2AA-Myc. Cells were grown in full DMEM media, fixed, labelled with antibodies for HA and ULK1 and imaged (Scale bar=10 µm). Quantification of overlap shown as percent of total Venus puncta (bottom). Mean ± SEM, n=1 independent experiment with 10 technical replicates. (D) HCT116 ATG9A-HA KI ATG13 KO cells reconstituted with stably expressing ATG13 WT, ATG13 Δ2AA and ATG13 ΔHORMA grown in full DMEM media, lysed and immunoblotted with indicated proteins. (E) Confocal images of EGFP-p62/SQSTM1 colocalization with HA ATG9A and ATG13 in HCT116 ATG9A-HA KI cells stably expressing EGFP-p62/SQSTM1. Cells were grown in full DMEM media with or without 1 μM Wortmannin for time 4 hrs, fixed, labelled with antibodies for HA and ATG13 and imaged (Scale bar=10 µm).
Aster-C Coordinates with COP I Vesicles to Regulate Lysosomal Trafficking and Activation of mTORC1 (2020)
Figure 1

Figure 1 Aster-C deficiency leads to hyper-activation of mTORC1.

A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min.

B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.

D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).

E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA.

F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.

G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).

H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.

Data information: Data are representative of at least three independent experiments.