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: Oncogene

Article Title: CBAP modulates Akt-dependent TSC2 phosphorylation to promote Rheb-mTORC1 signaling and growth of T-cell acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0507-6

Figure Lengend Snippet: CBAP deficiency reduces aerobic glycolysis and energy metabolism and attenuates the Raf-MEK-ERK–RSK and mTORC1-S6K-4E-BP1 signaling pathways. a Functional categories of KEGG gene sets markedly impacted (FDR < 0.25) by CBAP downregulation. b Correlation of CBAP positively-regulated genes and TSC1/TSC2-dependent rapamycin-sensitive genes , as determined by GSEA. c qRT-PCR analysis of the mRNA levels of genes involved in the regulation of glycolysis and fatty acid biosynthesis. Gapdh was used as an internal control. The data are averages of biologically triplicated experiments. Results are plotted as mean ± SD ( n = 3). d Expression of CBAP, c-Myc, P53 and HIF1α in Jurkat-derived cell clones. e Analysis of concentrations of lactate and α-ketoglutarate in the culture media of Jurkat and CBAP-KO cells. Data are expressed as means ± SD (n = 3; duplicate per measurement). f Suppression of p70S6K phosphorylation in cr-Ctrl and leukemic CBAP-KO Jurkat cells treated with the indicated doses of rapamycin for 6 h. Data represent mean ± SD of three independent experiments with duplicates of each condition. g Effect of the mTORC1 inhibitor, rapamycin, on Jurkat-derived leukemic cell growth. Data represent mean ± SD of three independent experiments. h , i Activation of signaling proteins involved in the Akt-TSC2-mTORC1 ( h ) and Raf-MEK-ERK ( i ) signaling pathways in the different Jurkat-derived cell lines, as indicated. Numbers under the lanes represent phosphor-proteins/total proteins normalized to that of cr-Ctrl cells (as 1.0). j , k Effects of the Akt inhibitor MK2206 ( j ) and the MEK inhibitor U0126 ( k ) on the growth of cr-Ctrl Jurkat and CBAP-KO leukemic Jurkat cells in tissue culture. Data represent mean ± SD of three independent experiments with duplicates of each condition. * P < 0.05; ** P < 0.01; *** P < 0.001, according to two-tailed unpaired Student's t tests

Article Snippet: Plasmids expressing Flag-tagged TSC2 (#14129) and Myc-tagged TSC1 (#12133) were obtained from Addgene (Cambridge, MA).

Techniques: Functional Assay, Quantitative RT-PCR, Expressing, Derivative Assay, Clone Assay, Activation Assay, Two Tailed Test

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

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: X ray is the CpG locus, and Y ray is the DNA methylation level from 0 (no methylation) to 1(100% methylation). Region 7 is located in SMAD1 region which is on Chromosome 17 from 12,487,811 to 12,488,083. Region 9 is located in TSC1 region which is on Chromosome 3 from 3925441 to 3925953. Region 10 is located in AKT1 region which is on Chromosome 18 from 67,877,536 to 67,878,025.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: DNA Methylation Assay, Methylation

Journal: Scientific Reports

Article Title: DNA methylation Landscape of body size variation in sheep

doi: 10.1038/srep13950

Figure Lengend Snippet: ( a ) RNA expression levels of BMPR1B , SMAD1 , SUMARF1 , TSC1 , and AKT1 . Interestingly, AKT1 showed significant level in UQ compared with StH and Tan. ( b ) Protein expression levels of BMPR1B, SMAD1, TSC1 and AKT1. There is no significant protein expression level observed among breeds. ( c ) TSC1 and TSC1 (Ser505) protein expression levels among UQ, Tan, and StH.

Article Snippet: Separated proteins were then transferred onto nitrocellulose membranes, which were incubated overnight with one of the following primary antibodies: BMPR1B(1:1000, 40–9400, Invitrogen), TSC1(1:1000, bs-3837R, Bioss), TSC1/Ser505 (1:1000, bs-5600R, Bioss), AKT1/Thr308 (1:1000, 2965, CST), SMURF1(1:1000, bs-9391R, Bioss), SMAD1(1:1000, 6944, CST), SMAD1/Ser465(1:500, sc-101800, Santa).

Techniques: RNA Expression, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-301a Suppression within Fibroblasts Limits the Progression of Fibrosis through the TSC1/mTOR Pathway

doi: 10.1016/j.omtn.2020.05.027

Figure Lengend Snippet: miR-301a Directly Targeted TSC1 in Fibroblasts (A) Prediction of major interference sites between miR-301a and the TSC1 mRNA 3′ UTR using TargetScan. (B) Luciferase activity in 293T cells transfected with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (C) Western blot analysis of TSC1 expression in 293T cells with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (D) Western blot analysis of Tsc1 and PTEN expression in MEFs isolated from WT or miR-301a −/− mice. (E) Western blot analysis of TSC1 and PTEN expression in HFL1 cells transfected with either anti-Ctl or anti-miR-301a. (F) Western blot analysis of TSC1 and PTEN expression in IPF fibroblasts transfected with either anti-Ctl or anti-miR-301a. (G) The expression of TSC1 in lung tissues from patients with IPF and normal donors, as determined by qPCR. (H) The expression of TSC1 in fibroblasts isolated from the lungs of patients with IPF lung and from nonfibrotic lungs of normal donors, as determined by qPCR. Values are expressed as mean ± standard deviation. ∗p < 0.05, for differences between the indicated groups.

Article Snippet: The primary antibodies were as follows: TSC1 (20988-1-1AP, Proteintech, USA), p-mTOR-S2448 (381557, ZenBio, USA), p-P70S6K1-T389 (AP0564, ABclonal, China), anti-Fn (ab2413, Abcam, USA), anti-α-SMA (ab5694, Abcam), vimentin (A11952, ABclonal), Ki67 (A2094, ABclonal), STAT3 (9139, CST, USA), p-Stat3-Tyr705 (9145, CST), PTEN (A11193, ABclonal), SMAD4 (38454, CST), TP63 (A12968, ABclonal), PIAS3 (A7060, ABclonal), and β-actin (AC026, ABclonal).

Techniques: Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Isolation, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-301a Suppression within Fibroblasts Limits the Progression of Fibrosis through the TSC1/mTOR Pathway

doi: 10.1016/j.omtn.2020.05.027

Figure Lengend Snippet: Inhibition of Lung Fibrosis in miR-301a −/− Mice Correlated with Inhibition of mTOR through Upregulating TSC1 in Fibroblasts (A) Ki67 staining showed cell proliferation in fibrotic lung tissues from WT (n = 5) and miR-301a −/− (n = 5) mice treated with bleomycin. Scale bars, 50 μm. (B) Ki67-positive cell counts in fibrotic lung tissue sections. (C) Western blot analysis of Tsc1 expression in lung tissues from WT and miR-301a −/− mice with the indicated treatment. (D) Immunohistochemical staining for Tsc1 in fibrotic lung tissues from WT (n = 5) and miR-301a −/− (n = 5) mice treated with bleomycin. Scale bars, 50 μm. (E) Western blot analysis of phosphorylated mTOR (p-mTOR) and phosphorylated 70S6K1 (p-P70S6K1) expression in lung tissues. (F) Western blot analysis of p-mTOR and p-P70S6K1 expression in MEFs isolated from WT or miR-301a −/− mice. (G) Western blot analysis of p-mTOR and p-P70S6K1 expression in HFL1 cells transfected with either anti-Ctl or anti-miR-301a. (H) Western blot analysis of p-mTOR and p-P70S6K1 expression in IPF fibroblasts transfected with either anti-Ctl or anti-miR-301a. (I) HFL1 cells were transfected with either anti-Ctl or anti-miR-301a for 48 h and treated with TGF-β (10 ng/mL) at the indicated times. Western blot analysis of p-mTOR, p-P70S6K1, and TSC1 expression in HFL1 cells. (J) IPF fibroblasts were transfected with anti-Ctl or anti-miR-301a for 48 h and treated with TGF-β (10 ng/mL) at the indicated times. The expression of p-mTOR, p-P70S6K1, and TSC1 was measured by western blot analysis. (K) IPF fibroblasts were transfected with anti-Ctl or anti-miR-301a for 48 h and treated with IL-6 (10 ng/mL) at the indicated times. The expression of p-mTOR and TSC1 was measured by western blot analysis. (L) IPF fibroblasts were transfected with the indicated shRNA, including anti-Ctl, anti-miR-301a, or shRNA-TSC1. The expression of p-mTOR and TSC1 was measured by western blot analysis. (M) IPF fibroblasts were transfected with the indicated plasmids for 24, 48, and 72 h, respectively. Cell proliferation was measured by a CCK-8 assay. Values are expressed as mean ± standard deviation. ∗∗p ≤ 0.01, for differences between WT and miR-301a −/− mice treated with bleomycin.

Article Snippet: The primary antibodies were as follows: TSC1 (20988-1-1AP, Proteintech, USA), p-mTOR-S2448 (381557, ZenBio, USA), p-P70S6K1-T389 (AP0564, ABclonal, China), anti-Fn (ab2413, Abcam, USA), anti-α-SMA (ab5694, Abcam), vimentin (A11952, ABclonal), Ki67 (A2094, ABclonal), STAT3 (9139, CST, USA), p-Stat3-Tyr705 (9145, CST), PTEN (A11193, ABclonal), SMAD4 (38454, CST), TP63 (A12968, ABclonal), PIAS3 (A7060, ABclonal), and β-actin (AC026, ABclonal).

Techniques: Inhibition, Staining, Western Blot, Expressing, Immunohistochemical staining, Isolation, Transfection, shRNA, CCK-8 Assay, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-301a Suppression within Fibroblasts Limits the Progression of Fibrosis through the TSC1/mTOR Pathway

doi: 10.1016/j.omtn.2020.05.027

Figure Lengend Snippet: Reducing Bleomycin-Induced Pulmonary Fibrosis in Mice Correlated with miR-301a Knockdown and Elevated Tsc1 Expression (A) Schematic representation of the tail vein injection with anti-Ctl or anti-miR-301a into WT mice, respectively (n = 5 per group). (B) The expression of miR-301a was evaluated by qPCR (n = 5 per group). (C) The expression of Tsc1 was detected by immunohistochemical analysis in WT mice. Scale bars, 75 μm. (D) H&E-stained sections and pathological scores of lung tissues. Scale bars, 100 μm. (E) Schematic representation of the tail vein injection with sh-Ctl or shTsc1 lentivirus supernatant into miR-301a −/− mice (n = 5 per group). (F) Tsc1 and p-mTOR expression in lung sections from miR-301a −/− mice (n = 3) with either sh-Ctl or shTsc1 injection was determined by western blot analysis. (G) The degree of fibrosis was shown with H&E staining (scale bars, 100 μm) and immunohistochemical analysis of α-SMA (scale bars, 50 μm) in lung tissues from miR-301a −/− mice. (H) Tsc1 and p-mTOR expression was measured by immunohistochemical staining. Scale bars, 50 μm. Values are expressed as mean ± standard deviation. ∗∗p < 0.01, for differences between the indicated groups.

Article Snippet: The primary antibodies were as follows: TSC1 (20988-1-1AP, Proteintech, USA), p-mTOR-S2448 (381557, ZenBio, USA), p-P70S6K1-T389 (AP0564, ABclonal, China), anti-Fn (ab2413, Abcam, USA), anti-α-SMA (ab5694, Abcam), vimentin (A11952, ABclonal), Ki67 (A2094, ABclonal), STAT3 (9139, CST, USA), p-Stat3-Tyr705 (9145, CST), PTEN (A11193, ABclonal), SMAD4 (38454, CST), TP63 (A12968, ABclonal), PIAS3 (A7060, ABclonal), and β-actin (AC026, ABclonal).

Techniques: Knockdown, Expressing, Injection, Immunohistochemical staining, Staining, Western Blot, Standard Deviation