Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Confirmation of Tsc1 mutant mice using Sanger sequencing. b Confirmation of Tsc2 mutant mice using Sanger sequencing. c MRI revealed imaging characteristics of the kidneys in 12 to 13 months mice (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). d Macroscopic appearance of 12 to 13 months mouse tissues (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). e Representative images of HE staining, SMA IHC staining, CD31 IHC staining, HMB45 IHC staining and PAS staining in liver tissues isolated from mice aged 12 to 13 months (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA − ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( e ). f Representative images of HE staining and PAS staining in kidney tissues isolated from mice aged 12 to 13 months (WT, Tsc1 c.2500-2503delAACA , Tsc2 c.1113delA ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( f ). Data are shown as the mean ± SD, n = 6, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Mutagenesis, Sequencing, Imaging, Staining, Immunohistochemistry, Isolation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Western blot (WB) analysis of mTORC1 activity in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ). b , c Analysis of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ) using Periodic Acid-Schiff (PAS) staining and a glycogen assay kit. Bar, 20 μm in ( b ). d Detection of mTORC1 activity and p-GSK3β(s9) levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) using WB at 24 h post treatment of 300 nM rapamycin. e , f Analysis of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) at 24 h post treatment of 300 nM rapamycin or control (DMSO) using PAS staining and glycogen assay kit. Bar, 20 μm in ( e ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Western Blot, Activity Assay, Staining, Control, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a , b Detection of m 6 A levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ) using an m 6 A quantification assay and m 6 A dot blot. c The main enzymes involved in m 6 A modification, created with Figdraw. d WB analysis of METTL3, METTL14, WTAP, FTO and ALKBH5 expression in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 ). e WB analysis of the expression of WTAP, METTL3 and p-P70S6 (Thr389) in MEFs (WT, Tsc1 −/− , Tsc2 −/− ) at 24 h post-treatment with 300 nM rapamycin or DMSO. f , g Detection of m 6 A levels in Tsc2 −/− MEFs (si-NC, si- Wtap- 1, si- Wtap- 2) using m 6 A quantification assay and m 6 A dot blot. h , i Detection of m 6 A levels in Tsc2 −/− MEFs (si-NC, si- Mettl3- 1, si- Mettl3- 2) using m 6 A quantification assay and m 6 A dot blot. Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Dot Blot, Modification, Expressing, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a WB analysis of mTORC1 activity, METTL3 and WTAP protein levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− , Tsc1/2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). b Detection of METTL3 expression in WT MEFs and HepG2 with transient overexpression of TSC1 or TSC2 . c Schematic representation of the presence of TSC1, TSC2, and TSC complex forms in four types of MEFs, created using Figdraw. TSC1 knockout ( Tsc1 –/– MEFs) causes loss of TSC1 and increased expression of uncomplexed-TSC2, due to loss of TSC1 leads to reductions of TSC2 at the same time, only increased a part of expression of uncomplexed-TSC2. TSC2 knockout ( Tsc2 –/– MEFs) causes the loss of TSC2 and increased uncomplexed-TSC1 level, double knockout ( Tsc1/2 –/– MEFs) caused loss of TSC1 and TSC2. d , e Analysis of TSC1, TSC2 and TSC complex in samples prepared from WT MEFs and HepG2 post s ucrose density-gradient centrifugation using WB (fractions 1 to 9 were arranged from top to bottom). f Analysis of mTORC1 activity in HepG2 with transient overexpression of TSC1 or TSC2 . g , h Analysis of glycogen levels in MEFs and HepG2 using PAS staining and the glycogen assay kit at 48 h post transient overexpression of TSC1 or TSC2 . Bar, 20 μm in ( g ). i , j Quantification of glycogen levels in MEFs (WT, Tsc1 −/− , Tsc2 −/− , Tsc1/2 −/− ) and HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). Bar, 20 μm in ( i ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Activity Assay, Expressing, Over Expression, Knock-Out, Double Knockout, Gradient Centrifugation, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Analysis of Mettl3 mRNA expression in all MEFs using qRT-PCR. b Analysis of METTL3 mRNA expression in oe-NC, oe- TSC1 and oe- TSC2 HepG2 using qRT-PCR. c Venn diagram showing overlaps between differential genes of three RNA-seq with GO molecular function including chromatin remodeling. d WB analysis of KDM5A in HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). e Analysis of KDM5A expression in MEFs (WT, Tsc1 −/− , Tsc 2 −/− , Tsc1/2 −/− ) at 24 h post treatment of 300 nM rapamycin or DMSO. f Analysis of KDM5A expression in oe-NC, oe- TSC1 and oe- TSC2 HepG2 using WB. g Detection of KDM5A and METTL3 in HepG2 at 48 h post transient overexpression or knockdown of KDM5A by transfection with overexpressing vector (oe- KDM5A ) or KDM5A -siRNA (si- KDM5A -1, si- KDM5A -2). h ChIP-PCR analysis of H3K4me3 modification within the METTL3 promoter in oe-NC and oe- KDM5A HepG2. i ChIP-PCR analysis of H3K4me3 modification within the METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. j ChIP-PCR analysis of KDM5A binding with METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. k ChIP-PCR analysis of TFs TBP, ETS1 and NRF1 binding with METTL3 promoter in oe-NC, oe- TSC1 and oe- TSC2 HepG2. l , m Detection of glycogen levels in HepG2 using PAS staining and the glycogen assay kit at 48 h post transfection with TSC1 overexpressing vector, TSC1 overexpressing vector + KDM5A overexpressing vector, or TSC1 overexpressing vector + KDM5A overexpressing vector + METTL3 overexpressing vector. Bar, 20 μm in ( l ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Expressing, Quantitative RT-PCR, RNA Sequencing, Over Expression, Knockdown, Transfection, Plasmid Preparation, Modification, Binding Assay, Staining, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a WB analysis of METTL3 and GYS2 in Tsc2 −/− MEFs (si- M3 -1, si- M3 -2 ) . b WB analysis of METTL3 and GYS2 in oe-NC and oe- M3 HepG2. c WB analysis of GYS2 in HepG2 (si-NC, si- TSC1 , si- TSC2 , si- TSC1 + TSC2 ). d Analysis of GYS2 in MEFs (WT, Tsc1 −/− , Tsc 2 −/− , Tsc1/2 −/− ) at 24 h post-treatment with 300 nM rapamycin or DMSO using WB. e , f Detection of METTL3 and GYS2 in HepG2 at 48 h post the transfection of si- TSC2 or oe- TSC1 , and si- METTL3 . g M 6 A methylation detection of GYS2 in oe-NC and oe- TSC1 HepG2. h M 6 A methylation detection of GYS2 in oe-NC and oe- M3 HepG2. i Detection of the mRNA half-life of GYS2 in oe-NC and oe- TSC1 HepG2. j Detection of the half-life of mRNA GYS2 in oe-NC and oe- M3 HepG2. k Detection of IGF2BP2 and GYS2 in HepG2 at 48 h post the transfection of si- TSC2 and si- IGF2BP2 . l Detection of the mRNA half-life of GYS2 in si-NC and si- IGF2BP2 . m WB quantification of knockdown efficacy of GYS2 in HepG2 (si- GYS2 -1, si- GYS2 -2 targeting human GYS2 ). n , o Analysis of glycogen levels in HepG2 at 48 h post transfection of oe- M3 and si- GYS2 ; Bar, 20 μm in ( n ). Data are shown as the mean ± SD, n = 3, two-tailed unpaired Student’s t -test.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Transfection, Methylation, Knockdown, Two Tailed Test

Journal: Cell Death & Disease

Article Title: Uncomplexed-TSC1 deploys novel mTORC1-independent pathway to exacerbate the liver glycogen storage in TSC

doi: 10.1038/s41419-025-08161-3

Figure Lengend Snippet: a Representative images of PAS staining in liver tissues isolated from mice aged 12 to 13 months (WT, Tsc1 +/− , Tsc2 +/− and Tsc1 +/− / Tsc2 +/− ). n = 6 for each genotype; each group contains mice from three different litters. Bar, 100 μm in ( a ). b Representative images of PAS staining from tumor of nude mice (WT, Tsc1 −/− , Tsc2 −/− ). n = 5 for each genotype. Bar, 50 μm in ( b ). c Representative images of METTL3 IHC staining in liver tissue isolated from mice aged 13 to 14 months and METTL3 IHC scores of all groups. n = 6 for each genotype; each group contains mice from three different litters. Bar, 50 μm in ( c ). d Representative images of GYS2 IHC staining in liver tissue isolated from mice aged 13 to 14 months and GYS2 IHC scores of all groups. n = 6 for each genotype; each group contains mice from three different litters. Bar, 50 μm in ( d ). e In vivo fluorescence imaging of all mice injected rAAV8, n = 5 for each group; each group contains mice from three different litters. f Representative images of HE staining, SMA IHC staining, CD31 IHC staining and PAS staining in liver tissues isolated from mice injected rAAV8 after 6 weeks. n = 5 for each group; each group contains mice from three different litters. Bar, 100 μm in ( f ). g Tumor sizes in each group at 20 days post drug treatment, n = 5 for each group. h The tumor volume in each group were monitored every 5 days starting from 1 day post drug treatment. n = 5 for each group. i The tumor weight in each group 20 days after drug treatment. j Representative images of PAS staining from tumor 20 days post drug treatment of nude mice. n = 5 for each group. Bar, 50 μm in j . k Schematic of the proposed mechanism, created by Figdraw. Data are shown as the mean ± SD, n = 5 ~ 6, two-way RM ANOVA and Tukey’s multiple comparisons test for ( h ), two-tailed unpaired Student’s t -test for others.

Article Snippet: The pcDNA3.1-myc- TSC1 and pcDNA3.1-Flag- TSC2 plasmids were preserved by our lab; pcDNA3.1- METTL3 -WT-3*HA was purchased from PPL (Jiangsu, China); pcDNA3.1- METTL3 -mut-3*HA were constructed in our Lab; pcDNA3.1- KDM5A -Flag purchased from Cyagen (Suzhou, China) for overexpression experiments.

Techniques: Staining, Isolation, Immunohistochemistry, In Vivo, Fluorescence, Imaging, Injection, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease

doi: 10.1016/j.jbc.2024.105641

Figure Lengend Snippet: Reagent resources and list of primers

Article Snippet: Rabbit monoclonal anti-Hamartin/TSC1 (D43E2) , Cell signaling , Cat#6935; RRID: AB_10860420.

Techniques: Electron Microscopy, Recombinant, Protease Inhibitor, Staining, Isolation, Clinical Proteomics, Membrane, Enzyme-linked Immunosorbent Assay, Activation Assay, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: miR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

doi: 10.1038/s41418-018-0243-z

Figure Lengend Snippet: Hypoxia downregulates TSC1. a Expression levels of components of the mTOR signaling pathway, such as mTOR, TSC1, TSC2 and RHEB, were compared in PASMCs exposed to normoxia or hypoxia by NGS-based RNA sequencing. b Expression levels of TSC1 mRNA normalized to 18S rRNA were examined by qRT-PCR in PASMCs exposed to normoxia or hypoxia for 8 h or 24 h. * p < 0.05. c PASMCs were treated with 25 ng/ml actinomycin D for 2 h prior to hypoxia exposure for 8 h or 24 h. Level of TSC1 transcripts relative to 18S rRNA was measured by qRT-PCR. * p < 0.05. d Total cell lysates from PASMCs exposed to normoxia or hypoxia for 24 h were subjected to immunoblot analysis with anti-TSC1 or anti-β-actin antibody. By densitometry, relative amounts of TSC1 protein normalized to β-actin were quantitated. * p < 0.05

Article Snippet: Expression plasmid of TSC1 transcript with deleted 3′-UTR, pcDNA3.1 myc TSC1 (Addgene plasmid # 12133), was a gift from Cheryl Walker [ , ].

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot

Journal: Cell Death and Differentiation

Article Title: miR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

doi: 10.1038/s41418-018-0243-z

Figure Lengend Snippet: Regulation of mTOR signaling by TSC1 affects VSMC proliferation. a (Left panel) Expression levels of TSC1 mRNA normalized to 18S rRNA were examined by qRT-PCR in PASMCs transfected with control or siRNA against TSC1 (siTSC1) for 24 h. * p < 0.05. (Right panel) Total cell lysates from PASMCs transfected with control or siTSC1 for 24 h were subjected to immunoblot analysis with antibody against TSC1, pS6K1, S6K1 or β-actin. By densitometry, relative amounts of TSC1 protein normalized to β-actin and phosphorylated S6K1 protein normalized to total S6K1 were quantitated. * p < 0.05. b Representative images of Ki-67 immunostaining of PASMCs transfected with control or siTSC1, and calculation of Ki-67 index. Approximately 200 cells from at least 10 independent fields were counted for each condition, and Ki-67-positive cells are presented as a percentage of the total population. Scale bar represents 50 μm. * p < 0.05

Article Snippet: Expression plasmid of TSC1 transcript with deleted 3′-UTR, pcDNA3.1 myc TSC1 (Addgene plasmid # 12133), was a gift from Cheryl Walker [ , ].

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Western Blot, Immunostaining

Journal: Cell Death and Differentiation

Article Title: miR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

doi: 10.1038/s41418-018-0243-z

Figure Lengend Snippet: Hypoxia regulates the expression level of miR-92b-3p. a The miR-92b-3p MRE sequence found in the 3′-UTR of TSC1 mRNA is evolutionarily conserved. Hsa: human, Mmu: mouse, Rno: rat, Ssc: pig. b . Expression levels of miR-92b-3p normalized to U6 snRNA were examined by qRT-PCR in PASMCs exposed to normoxia or hypoxia for 24 h. * p < 0.05. c pri- and pre-miR-92b-3p levels relative to 18S rRNA and mature miR-92b-3p levels relative to U6 snRNA were measured by qRT-PCR at 8 h and 24 h after exposure to hypoxia. Data represent fold change of the RNA levels in hypoxia-exposed PASMCs from normoxia control cells. * p < 0.05

Article Snippet: Expression plasmid of TSC1 transcript with deleted 3′-UTR, pcDNA3.1 myc TSC1 (Addgene plasmid # 12133), was a gift from Cheryl Walker [ , ].

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Control

Journal: Cell Death and Differentiation

Article Title: miR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

doi: 10.1038/s41418-018-0243-z

Figure Lengend Snippet: TSC1 is a target of miR-92b-3p. a (Left panel) Schematic diagram of predicted miR-92b-3p MRE in the 3′-UTR of TSC1 transcripts and luciferase reporter constructs used for luciferase assays. CDS and AAA stand for protein coding sequence and poly(A) tail, respectively. Mutations introduced in the MRE to disrupt a base paring with miR-92b-3p sequence are indicated as black circles. (Right panel) Sequences of the 3′-UTR, the 3′-UTR mt, the wild-type miR-92b-3p MRE in the 3′-UTR of TSC1 (MRE) and the MRE mt cloned into the 3′-UTR of the luciferase gene are shown. The mutated sequence is in bold. Perfect base matches are indicated by a line. b Luciferase activity of constructs, such as the 3′-UTR, the 3′-UTR mt, the MRE and MRT mt, were examined in Cos7 cells by transfecting control or miR-92b-3p mimic. A luciferase vector without 3′-UTR sequence (Vector) was used as a negative control. Data represent the mean ± S.E. of triplicates. * p < 0.05. c . Levels of endogenous TSC1 mRNA relative to 18S rRNA were quantified by qRT-PCR analysis in PASMCs transfected with control mimic, miR-92b-3p mimic, or anti-miR-92b-3p. Data represent the mean ± S.E. of triplicates. * p < 0.05. d Immunoblot analysis of TSC1 and β-actin using cell lysates of PASMCs transfected with control mimic, miR-92b-3p mimic or anti-miR-92b-3p were performed. Protein bands were quantitated by densitometry, and relative amounts of protein normalized to β-actin are presented. * p < 0.05. e Levels of endogenous miR-92b-3p relative to U6 snRNA were quantified by qRT-PCR analysis in PASMCs transfected with control mimic, miR-92b-3p mimic, or anti-miR-92b-3p. Data represent the mean ± S.E. of triplicates. * p < 0.05

Article Snippet: Expression plasmid of TSC1 transcript with deleted 3′-UTR, pcDNA3.1 myc TSC1 (Addgene plasmid # 12133), was a gift from Cheryl Walker [ , ].

Techniques: Luciferase, Construct, Sequencing, Clone Assay, Activity Assay, Control, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Transfection, Western Blot

Journal: Cell Death and Differentiation

Article Title: miR-92b-3p-TSC1 axis is critical for mTOR signaling-mediated vascular smooth muscle cell proliferation induced by hypoxia

doi: 10.1038/s41418-018-0243-z

Figure Lengend Snippet: miR-92b-3p-mediated inhibition of TSC1 modulates mTOR signaling. PASMCs transfected with TSC1-expressing vector containing the miR-92b-3p binding site (Control) or TSC1 expression construct carrying the TSC1 cDNA deleted in the 3′-UTR (TSC1 wo3 ′ UTR ) were exposed to normoxia or hypoxia for 24 h. Total cell lysates were subjected to immunoblot analysis with antibody against TSC1, pS6K1, S6K1 or β-actin. By densitometry, relative amounts of TSC1 protein normalized to β-actin and phosphorylated S6K1 protein normalized to total S6K1 were quantitated. * p < 0.05

Article Snippet: Expression plasmid of TSC1 transcript with deleted 3′-UTR, pcDNA3.1 myc TSC1 (Addgene plasmid # 12133), was a gift from Cheryl Walker [ , ].

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Binding Assay, Control, Construct, Western Blot

Journal: Skeletal Muscle

Article Title: Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

doi: 10.1186/2044-5040-3-6

Figure Lengend Snippet: Conditional inactivation of TSC1 in skeletal muscle. ( A ) Western blot analysis of soleus muscle from 90-day-old control (ctrl) and TSCmKO mice using antibodies directed against the proteins indicated. α-actinin is used as a loading control. ( B ) Weight of soleus (Sol), gastrocnemius (GC), plantaris (PL), tibialis anterior (TA), extensor digitorum longus (EDL) and triceps (Tri) muscles of TSCmKO and littermate control (ctrl) mice. Weight is expressed as a percentage of the weight of the same muscle in control mice after normalization to the total body weight (N = 8 to 12 mice for each genotype). Data are mean ± SEM; *** P <0.001; ** P <0.01; * P <0.05; Student’s t -test. ( C ) H&E staining of cross-sections from TA and soleus muscles of control and TSCmKO mice. Scale bar = 50 μm. ( D, E ) Fiber size distribution in soleus ( D ) and TA ( E ) muscles of 90-day-old TSCmKO and control mice (N = 4). More details of fiber size analysis are shown in Additional file : Figure S3 and in Additional file , Table S1. * P <0.05.

Article Snippet: The antibodies used were from the following sources: rabbit polyclonal antibodies directed to 4E-BP1 (Phas-I) from Zymed (Life Technologies); those recognizing Phospho-4E-BP1 (Ser65), PKB/Akt, mTOR, S6 Ribosomal Protein or Phospho-S6 Ribosomal Protein (Ser235/236) were all from Cell Signaling Technology Inc. (Danvers, MA, USA); those against FoxO1a were from Abcam plc. (Cambridge, UK); those against TSC1 were from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Western Blot, Control, Muscles, Staining

Journal: Skeletal Muscle

Article Title: Differential response of skeletal muscles to mTORC1 signaling during atrophy and hypertrophy

doi: 10.1186/2044-5040-3-6

Figure Lengend Snippet: TSC1-raptor double knockouts resemble RAmKO mice. ( A ) Western blot analysis of soleus muscles of TSCmKO, TSC-RAmKO and control (ctrl) mice using antibodies directed against the proteins indicated. An equal amount of protein was loaded in each lane. Loading control was α-actinin. ( B ) Muscle weight of the tibialis anterior (TA) and soleus (Sol) muscles of TSC-RAmKO and control mice. Muscle weight was first normalized to the body weight and is expressed as percentage of the weight of the same muscle from control mice (N = 3 mice for each genotype). ( C ) Relative mRNA expression of Pgc1α and Pgc1β in soleus muscle of TSC-RAmKO and ctrl mice. Values obtained in control mice were set to 100% (N = 3 mice). Bars in B and C represent mean ± SEM. P -values are *** P <0.001; ** P <0.01; * P <0.05. ( D ) NADH-TR staining of soleus muscle from TSC-RAmKO and control mice. Scale bar = 100 μm. ( E ) Schematic drawing of the major signaling pathways regulated by mTORC1 and their influence on protein synthesis and degradation.

Article Snippet: The antibodies used were from the following sources: rabbit polyclonal antibodies directed to 4E-BP1 (Phas-I) from Zymed (Life Technologies); those recognizing Phospho-4E-BP1 (Ser65), PKB/Akt, mTOR, S6 Ribosomal Protein or Phospho-S6 Ribosomal Protein (Ser235/236) were all from Cell Signaling Technology Inc. (Danvers, MA, USA); those against FoxO1a were from Abcam plc. (Cambridge, UK); those against TSC1 were from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Western Blot, Muscles, Control, Expressing, Staining, Protein-Protein interactions

Journal: bioRxiv

Article Title: mTORC1-Plin3 pathway is essential to activate lipophagy and protects against hepatosteatosis

doi: 10.1101/812990

Figure Lengend Snippet: A) Levels of mTOR pathway proteins in total homogenates and LD isolations at 6 hours of OA treatment. Active mTOR, Raptor, Rheb and RagA, B and C, but not Rictor or TSC1, accumulate in LDs after lysosomal inhibition. B-C) mTOR (magenta) recruitment to LDs (green) is enhanced after blocking autophagy in the OA-treated primary hepatocytes (B, quantified in C). D-E) mTOR recruitment is also enhanced after blocking autophagy through Atg7 silencing in OA-treated NIH-3T3 cells. (D quantified in E). Scale bar: 20μm. Bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 (differences caused by lysosomal inhibitors treatment), # p<0.05, ## p<0.01, and ### p<0.001 (differences caused by treatment)

Article Snippet: Antibodies for Atg16l (PM040) from MBL; Atg7 (2631), Beclin 1 (3495), FIP200 (12436), LC3B (2775), mTOR (2983), phopho-mTOR (5536), Rag A (4357), Rag C (3360), Raptor (2280), Rictor (2114), S6 (2217), phopho-S6 (4858), TSC1 (6935) and anti-rabbit (7074) were from Cell Signaling Technology; RagB (NBP1-85801) from Novus Biologicals; Plin3 (3883,for WB) from ProSci; LAMP1 (1D4B, for WB) from Developmental Studies Hybridoma Bank; PLIN3 (GP30 for CoIP) from ProGen Biotechnik; β-actin (A5441) and anti-mouse (AP130P) and anti-rat (AP136P) from SIGMA; GAPDH (ab8245), Lamp1 (ab24170 for IF) and normal IgG (ab188776) from Abcam; Fluorescence secondary antibodies (Alexa Fluor 488 and/or Alexa Fluor 647 conjugated) and anti-guinea pig (A18775) from Fisher Scientific and Beclin 1-HRP(sc-48341 HRP) and Plin 3-HRP (sc-390968 HRP) form Santa Cruz Biotechnology.

Techniques: Inhibition, Blocking Assay