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: 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: 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

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: Inflammatory-miR-301a circuitry drives mTOR and Stat3-dependent PSC activation in chronic pancreatitis and PanIN

doi: 10.1016/j.omtn.2022.01.011

Figure Lengend Snippet: Inhibition of PanIN formation in miR-301a −/− ; Pdx1-Cre ; Kras G12D mice correlates with elevated Gadd45g expression and reduced PSCs proliferation (A) Venn diagram of specific genes between predicted miR-301a targets from miRWalk database and DEGs identified by RNA-seq. (B) Real-time PCR validation of 17 significantly upregulated genes from (A). (C) Prediction of major interference sites between miR-301a and the Gadd45g mRNA 3′ UTR using TargetScan. (D) Luciferase activity in 293T cells transfected with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (E) Western blot analysis of Gadd45g expression in 293T cells with the indicated luciferase reporter with either a control plasmid or a precursor miR-301a plasmid. (F) Western blot analysis of Gadd45g and Tsc1 expression in human PSCs transfected with anti-control (Anti-Ctl) or LNA-anti-miR-301a (Anti-miR-301a) oligonucleotide. (G) Western blot analysis of Gadd45g and Tsc1 expression in human PSCs transfected with anti-control (Anti-Ctl) or LNA-anti-miR-301a (Anti-miR-301a) oligonucleotide. (H) Expression of miR-301a in hPSCs treated with IL-6 or TGF-β. (I) Expression of miR-301a in mouse embryonic fibroblasts (MEFs), mouse PSCs (mPSCs), or Pan02 cells treated with IL-6 or TGF-β. (J) Cell viability of WT and miR-301a −/− PSCs treated with TGF-β. (K) Cell viability of miR-301a −/− PSCs transfected with shRNA-control (shCtl) or shRNA-Gadd45g (shGadd45g) and treated with TGF-β at the indicated time. Values are mean ± SD. ∗∗p < 0.01, ∗p < 0.05 indicate significant difference between the indicated groups. NS, not significant.

Article Snippet: The primary antibodies used were Tsc1 (BA2879, BOSTER), phospho-mTOR-S2448 (381557, ZENBIO), Fibronectin (ab2413, Abcam), α-SMA (ab5694, Abcam), Gadd45g (A10286, Abclonal), Stat3 (9139, CST), p-Stat3-Tyr705 (9145, CST), β-actin (AC026, Abclonal).

Techniques: Inhibition, Expressing, RNA Sequencing, Real-time Polymerase Chain Reaction, Biomarker Discovery, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Western Blot, shRNA