mouse: floxed raptor  (Jackson Laboratory)

Journal: Molecular Biology of the Cell

Article Title: TSC2-mTORC1 axis regulates morphogenesis and neurological function of Gli1 + adult-born dentate granule cells

doi: 10.1091/mbc.E24-08-0366

Figure Lengend Snippet: Genetic Raptor reduction reverses mTORC1 hyperactivation and neuronal hypertrophy in Tsc2 KO hippocampal cultures. (A) Schematic of the experimental protocol. WB: Western blotting; ICC: immunocytochemistry. (B) ICC data show TSC2 protein expression lost in GFP + primary hippocampal neurons carrying the Tsc2 flox/flox allele. Ai9 + cells with no TSC2 coexpression in magenta and Ai9 + cells with TSC2 coexpression in white. (C) Quantification showing the loss of TSC2 protein expression in ∼50% of hippocampal neurons. Data were obtained from three independent infection experiments. One-way ANOVA, multiple comparisons, Sidak. Means: Control = 92% ± 1.155%, Tsc2 KO = 42.333% ± 2.728%, and Tsc2 KO ; Raptor ± = 41.333% ± 5.548%, Tsc2 KO ; Rictor ± = 51.667% ± 7.446%. (D) Representative WB data from Ctrl ( Tsc2 flox/flox hippocampal culture treated with AAV-eGFP), Tsc2 KO ( Tsc2 flox/flox hippocampal culture treated with AAV-CRE-eGFP), Tsc2 KO ; Raptor ± ( Tsc2 flox/flox ;Raptor flox/+ hippocampal culture treated with AAV-CRE-eGFP), and Tsc2 KO ; Rictor ± ( Tsc2 flox/flox ;Rictor flox/+ hippocampal culture treated with AAV-CRE-eGFP). RAPTOR signals were obtained from 8% SDS–PAGE, and the others from 12% SDS–PAGE. (E–I) Bar graphs illustrating the quantification of mTORC1 and mTORC2 pathway members shown as a relative fold-change of control levels (mean ± SEM). Each dot represents one independent sample, three independent samples were used. One-way ANOVA, multiple comparisons, Tukey. Means: RAPTOR-Control = 1 ± 0, Tsc2 KO = 1.173 ± 0.086, Tsc2 KO ; Raptor ± = 0.535 ± 0.036, Tsc2 KO ; Rictor ± = 1.172 ± 0.166; RICTOR-Control = 1 ± 0, Tsc2 KO = 0.881 ± 0.092, Tsc2 KO ; Raptor ± = 0.647 ± 0.024, Tsc2 KO ; Rictor ± = 0.375 ± 0.042; pS6-Control = 1 ± 0, Tsc2 KO = 1.817 ± 0.092, Tsc2 KO ; Raptor ± = 0.619 ± 0.194, Tsc2 KO ; Rictor ± = 1.403 ± 0.138; p-AKT-Control = 1 ± 0, Tsc2 KO = 0.775 ± 0.097, Tsc2 KO ; Raptor ± = 0.760 ± 0.042, Tsc2 KO ; Rictor ± = 0.596 ± 0.085; p-4EBP-Control = 1 ± 0, Tsc2 KO = 1.010 ± 0.067, Tsc2 KO ; Raptor ± = 0.958 ± 0.203, Tsc2 KO ; Rictor ± = 0.799 ± 0.137. (J) Representative images of eGFP-expressing hippocampal neurons (left) with Neurolucida reconstructed soma surface area (right). Scale bars = 10 µm. (K) Bar graphs indicating the soma surface area of hippocampal neurons. Each dot represents one neuron, an equivalent number of neurons analyzed from three independent infection experiments. Sixty control cells, 60 Tsc2 KO cells, 60 Tsc2 KO ; Raptor ± cells, and 60 Tsc2 KO ; Rictor ± cells. One-way ANOVA, multiple comparisons, Sidak. Means: Control = 343.956 µm 2 ±16.973 µm 2 , Tsc2 KO = 559.333 µm 2 ±25.491 µm 2 , Tsc2 KO ; Raptor ± = 435.182 µm 2 ±26.116 µm 2 , Tsc2 KO ; Rictor ± = 627.244 µm 2 ±28.842 µm 2 . ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Tsc2 flox/flox , Gli1 CreERT2 , Rosa Tdt , Thy1-GFP , Rictor flox/flox , and Raptor flox/flox mice were obtained from The Jackson Laboratory.

Techniques: Western Blot, Immunocytochemistry, Expressing, Infection, Control, SDS Page

Journal: Molecular Biology of the Cell

Article Title: TSC2-mTORC1 axis regulates morphogenesis and neurological function of Gli1 + adult-born dentate granule cells

doi: 10.1091/mbc.E24-08-0366

Figure Lengend Snippet: Genetic inhibition of mTORC1 reverses mTORC1 hyperactivation, hypervascularization, increased cellular senescence, and reduced NEUN expression in Tsc2 KO mice. (A) A representative image of labeling of Gli1 + dGCs in the DG region of Gli1 CreERT2 ; Rosa Ai9/+ mice (Ai9 + cells in magenta) 6 wk after a single TAM injection at postnatal (P) day 14. Scale bars = 100 µm. (B) Representative images of TSC2 protein expression in the Gli1 + dGCs of Tsc2 KO mice ( Gli1 CreERT2 ; Tsc2 flox/flox ; Rosa Td/+ ). The percentage of Ai9 + dGCs that express TSC2 is quantified on the right. Each dot represents one mouse, six tissue sections analyzed per mouse. Yellow arrowheads indicate Ai9 + cells (Gli1 + adult-born dGCs, magenta) that coexpress TSC2 while white arrowheads indicate Ai9 + cells that lack coexpression of TSC2. Unpaired t test. Means: Control = 85.048 ± 0.995, Tsc2 KO = 55.303 ± 1.623. Scale bars = 10 µm. (C–D) Expression of p-S6 in Ai9 + dGCs. Each dot represents one mouse, six tissue sections analyzed per mouse. Yellow arrowheads indicate p-S6 expression. Control = 1.0 ± 0.024, Tsc2 KO = 1.433 ± 0.082, Tsc2 KO ; Raptor ± = 0.985 ± 0.065. One-way ANOVA, multiple comparisons, Tukey. Scale bars = 10 µm (E) Representative images of blood vessels immunostained with CD31 surrounding Ai9 + dGCs in control, Tsc2 KO , and Tsc2 KO ; Raptor ± mice (yellow squares are magnifications). Yellow arrowheads denote CD31 expression. Scale bars = 10 µm. (F) Quantification of DG CD31 + blood vessels in Tsc2 KO mice. Each dot represents one mouse, and six tissue sections were analyzed per mouse. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 0.0003 ± 0.00001, Tsc2 KO = 0.0003 ± 0.00001, and Tsc2 KO ; Raptor ± = 0.0002 ± 0.00003. (G) Representative images of SA-β-gal expression in control, Tsc2 KO , and Tsc2 KO ; Raptor ± mice (yellow squares are magnifications). Yellow arrowheads denote SA-β-gal expression. Scale bars = 10 µm. (H) Quantification of SA-β-gal in Tsc2 KO mice. Each dot represents average SA-β-gal expression from one mouse, six tissue sections analyzed per mouse. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 1.000 ± 0.035, Tsc2 KO = 1.364 ± 0.086, and Tsc2 KO ; Raptor ± = 0.868 ± 0.018. (I) Representative images showing Ai9 + dGCs immunostained with DCX in control and Tsc2 KO mice at 6 wk after TAM injection. White arrowheads indicate Ai9 + cells (Gli1 + adult-born dGCs, magenta) that lack coexpression of DCX. Scale bars = 10 µm. (J) Quantification of the percentage of Ai9 + dGCs that express DCX in control mice ( Gli1 CreERT2 ; Rosa Td/+ ) and Tsc2 KO mice ( Gli1 CreERT2 ; Tsc2 flox/flox ; Rosa Td/+ ) at 6 wk after TAM injection. Each dot represents one mouse, and six tissue sections were analyzed per mouse. Unpaired t test. Means: Control = 30.963 ± 1.858, Tsc2 KO = 34.004 ± 5.538. (K–L) Representative images (K) and bar graphs (L) of NEUN in Ai9 + dGCs of Tsc2 KO mice and Tsc2 KO ; Raptor ± mice ( Gli1 CreERT2 ; Tsc2 flox/flox ; Raptor flox/+ ; Rosa Td/+ ). Yellow arrowheads indicate Ai9 + cells (Gli1 + adult-born dGCs, magenta) that coexpress NEUN while white arrowheads indicate Ai9 + cells that lack coexpression of NEUN. Each dot represents one mouse, six tissue sections analyzed per mouse. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 75.556 ± 3.879, Tsc2 KO = 55.150 ± 0.621, and Tsc2 KO ; Raptor ± = 84.916 ± 0.455. Scale bars = 10 µm. ns, not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: Tsc2 flox/flox , Gli1 CreERT2 , Rosa Tdt , Thy1-GFP , Rictor flox/flox , and Raptor flox/flox mice were obtained from The Jackson Laboratory.

Techniques: Inhibition, Expressing, Labeling, Injection, Control

Journal: Molecular Biology of the Cell

Article Title: TSC2-mTORC1 axis regulates morphogenesis and neurological function of Gli1 + adult-born dentate granule cells

doi: 10.1091/mbc.E24-08-0366

Figure Lengend Snippet: Genetic inhibition of mTORC1 corrects multiple dendritic morphological defects in Tsc2 KO mice. (A) Representative images showing GFP-expressing control dGCs and Tsc2 KO dGCs in Tsc2 KO mice ( Gli1 CreERT2 ; Tsc2 flox/flox ; Thy1 GFP ) and Tsc2 KO ; Raptor ± dGCs in Tsc2 KO ; Raptor ± mice ( Gli1 CreERT2 ; Tsc2 flox/flox ; Raptor flox/+ ; Thy1 GFP ). Top: images showing dGCs immunostained with TSC2, bottom: Neurolucida reconstruction of somas. Yellow arrowheads indicate Ai9 + cells (Gli1 + adult-born dGCs, magenta) that coexpress TSC2 while white arrowheads indicate Ai9 + cells that lack coexpression of TSC2. Confocal z-series stacks were imported into Neurolucida for analysis. Scale bars = 10 µm. (B) Quantification of soma surface area in Tsc2 KO dGCs. An equivalent number of somas was analyzed from tissue sections of 3 mice per each group. Seventy-five control cells, 75 Tsc2 KO cells, and 51 Tsc2 KO ; Raptor ± cells. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 372.203 µm 2 ±14.294 µm 2 , Tsc2 KO = 644.121 µm 2 ±30.782 µm 2 , and Tsc2 KO ; Raptor ± = 332.876 µm 2 ±11.376 µm 2 . (C) Neurolucida reconstructions of GFP-expressing control, Tsc2 KO , and Tsc2 KO ; Raptor ± dGCs. (D) Quantification of total dendritic length in Tsc2 KO dGCs. An equivalent number of neurons were reconstructed from tissue sections of 3 mice per each group. Forty-one control cells, 41 Tsc2 KO cells, and 36 Tsc2 KO ; Raptor ± cells. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 406.282 µm ± 17.155 µm, Tsc2 KO = 602.474 µm ± 25.147 µm, and Tsc2 KO ; Raptor ± = 414.006 µm ± 8.981 µm. (E) Sholl analysis of Neurolucida reconstructions of GFP-expressing dGCs from control mice, Tsc2 KO , and Tsc2 KO ; Raptor ± dGCs. (F) Quantification of intersections in Tsc2 KO dGCs. An equivalent number of neurons were reconstructed from tissue sections of 3 mice per each group. Forty-one control cells, 41 Tsc2 KO cells, and 36 Tsc2 KO ; Raptor ± cells. Mixed-effects analysis, Tukey. Means: 50 µm Control = 2.927 (±0.176), Tsc2 KO = 3.854 (±0.248). 60 µm Control = 3.073 (±0.183), Tsc2 KO = 4.000 (±0.247). 80 µm Control = 2.902 (±0.181), Tsc2 KO = 3.951 (±0.212). 90 µm Control = 2.683 (±0.189), Tsc2 KO = 3.829 (±0.203). 100 µm Control = 2.390 (±0.188), Tsc2 KO = 3.756 (±0.212). 110 µm Control = 2.146 (±0.196), Tsc2 KO = 3.585 (±0.204). 120 µm Control = 1.732 (±0.168), Tsc2 KO = 3.439 (±0.218). 130 µm Control = 1.439 (±0.160), Tsc2 KO = 3.073 (±0.238). 140 µm Control = 1.244 (±0.151), Tsc2 KO = 2.683 (±0.196). 150 µm Control = 1.049 (±0.126), Tsc2 KO = 2.268 (±0.207). 160 µm Control = 0.951 (±0.121), Tsc2 KO = 1.927 (±0.183). (G) Convex hull analysis of Neurolucida reconstructions of GFP-expressing control, Tsc2 KO , and Tsc2 KO ; Raptor ± dGCs. (H) Quantification of total volume in Tsc2 KO dGCs. An equivalent number of neurons were reconstructed from tissue sections of 3 mice per each group. Forty-one control cells, 41 Tsc2 KO cells, and 36 Tsc2 KO ; Raptor ± cells. One-way ANOVA, multiple comparisons, Tukey. Means: Control = 102865.342 µm 3 ±9348.966 µm 3 , Tsc2 KO = 166442.330 µm 3 ±10277.416 µm 3 , and Tsc2 KO ; Raptor ± = 75681.583 µm 3 ±4384.555 µm 3 . ns, not significantly different, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Tsc2 flox/flox , Gli1 CreERT2 , Rosa Tdt , Thy1-GFP , Rictor flox/flox , and Raptor flox/flox mice were obtained from The Jackson Laboratory.

Techniques: Inhibition, Expressing, Control

Journal: Bone Research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: Constitutive activation of mTORC1 in preosteoblasts induces aberrant bone formation in subchondral bone and promotes OA in mice. a , b Representative photomicrographs and quantification of immunostaining of p-S6 in Osterix + preosteoblasts in tibial subchondral bone of transgenic mice after Tsc1 deletion (ΔTSC1) compared to that of their littermates (Ctrl). c , d Representative photomicrographs and quantification of immunostaining of Ki-67 in Osterix + preosteoblasts in tibial subchondral bone of transgenic mice after Tsc1 deletion (ΔTSC1) compared to that of their littermates (Ctrl). Boxed area is magnified on the top corner. Scale bars, 100μm. AC, articular cartilage; SCB, subchondral bone; GP, growth plate. e Representative Safranin O-Fast green and H&E staining of sagittal sections of articular cartilage and tibia subchondral bone of ΔTSC1 sham mice and ΔTSC1 ACLT mice compared to that of their littermates (Ctrl). Scale bars, 100 μm. f Representative 3D reconstructed micro-CT images of sagittal of tibia subchondral bone of ΔTSC1 sham mice and ΔTSC1 ACLT mice compared to that of their littermates (Ctrl). Scale bars, 1mm. g , h Quantitative analysis of bone mass in subchondral bone: bone volume/total volume (BV/TV) and bone mineral density (BMD). i OARSI scores based on the histology analysis. Data are shown as mean ± s.d. and analyzed by Student’s t test or two-way ANOVA . n ≥ 6, * P < 0.05, and ** P < 0.01

Article Snippet: We purchased the Tsc1 flox/flox , Raptor flox/flox , R26-mT/mG, and Osx1-GFP::Cre mouse strains from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Activation Assay, Immunostaining, Transgenic Assay, Staining, Micro-CT

Journal: Bone Research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: mTORC1 activation in subchondral preosteoblasts produce Cxcl12 to promote chondrocyte hypertrophic differentiation MSCs osteogenic differentiation. a Western blot and quantitative PCR analysis of Cxcl12 in primary preosteoblasts. b – d ) Cxcl12 assessed by ELISA in the cultured medium of primary preosteoblasts or in the serum at 6 weeks post ACLT surgery. e Representative immunostaining and quantitative analysis of CXCR4 + cells in articular cartilage and subchondral bone in ΔTSC1, ΔRaptor mice, and their littermates (Ctrl). Scale bars, 100 μm. Quantitative analysis of CXCR4 + cells out of total cartilage chondrocytes. f , g Quantitative PCR analysis of Col2, ACAN, Col X, MMP-13 mRNA in ADTC5 cells treated with the cultured medium of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. h , i Western blot analysis of Col2, Col X, MMP-13, and Toluidine blue staining in ADTC5 cells treated with CM of primary preosteoblasts and Cxcl12-neutralizing antibody for 7 days. j Western blot analysis of Col 1α, Col X, MMP-13, and Alizarin red staining in ADTC5 cells treated with CM of primary preosteoblasts, recombinant murine Cxcl12 or Cxcl12-neutralizing antibody for 14 days. Data are shown as mean ± s.d. and analyzed by Student’s t test and one-way ANOVA . n ≥ 8, ** P < 0.01

Article Snippet: We purchased the Tsc1 flox/flox , Raptor flox/flox , R26-mT/mG, and Osx1-GFP::Cre mouse strains from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Activation Assay, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunostaining, Staining, Recombinant

Journal: Bone Research

Article Title: Activation of mTORC1 in subchondral bone preosteoblasts promotes osteoarthritis by stimulating bone sclerosis and secretion of CXCL12

doi: 10.1038/s41413-018-0041-8

Figure Lengend Snippet: Administration of anti-Cxcl12 antibody attenuated subchondral bone remodeling and articular cartilage degeneration in ACLT mice. a , b , d , e Representative immunostaining and quantitative analysis of Cxcl12 in Osterix + preosteoblasts in the subchondral bone and metaphysis in ΔTSC1, ΔRaptor mice, and their littermates (Ctrl). Boxed area is magnified on the corner. Scale bar, 100 μm. AC, articular cartilage; SCB, subchondral bone; GP, growth plate; BM, bone marrow. c Representative Safranin O-Fast green, H&E staining, 3D reconstructed μ-CT images and immunohistochemical staining of OCN, type X collagen, and MMP-13 in ΔTSC1 mice administered with anti-Cxcl12 antibody or normal IgG for 6 weeks. Scale bar, 100 μm (Safranin O, H&E staining, OCN, type X collagen, and MMP-13), 1 mm (μ-CT). f OARSI scores based on the histological analysis. g , h Quantitative analysis of type X collagen and MMP-13 in articular cartilage. i , j Quantitative analysis of bone mass in subchondral bone: bone volume/total volume (BV/TV) and bone mineral density (BMD). k Quantitative analysis of OCN + osteoblasts in tibial subchondral bone. Data are shown as mean±s.d. and analyzed by Student’s t test. n ≥ 6, ** P < 0.01

Article Snippet: We purchased the Tsc1 flox/flox , Raptor flox/flox , R26-mT/mG, and Osx1-GFP::Cre mouse strains from the Jackson Laboratory (Bar Harbor, ME, USA).

Techniques: Immunostaining, Staining, Immunohistochemical staining