Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 1. Altering endogenous retinal and tectal BDNF levels in vivo. Diagrams representing a transverse view of a Xenopus tadpole brain and eye illustrate experimental procedures (see Materials and Methods). RGCs are depicted in red, relative endogenous BDNF expression levels (Cohen-Cory et al., 1996) are depicted in blue, and exogenously applied factors are depicted in green. A, Effects of altered tectal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated green fluorescent microspheres were injected into the stage 38 tadpole tectum. At stage 42, RGCs were retrogradely labeled by injecting rhodamine–dextran in the contralateral tectum. At stage 45, dendritic morphologies of double-labeled RGCs were evaluated. A low-power view of a tadpole eye shows green fluorescent microspheres retrogradely transported to the retinal ganglion cell layer, where a rhodamine–dextran-labeled RGC soma can also be visualized (lines denote lens and eye periphery). Scale bar, 50 m. A single-plane, high-power view of a stage 45 retina reveals a rhodamine–dextran-labeled RGC with internalized green fluorescent microspheres. Scale bar, 5 m. B, Effects of altered retinal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 38 tadpole retina, and then RGCs were retrogradely labeled at stage 42. The low-power view shows rhodamine–dextran-labeled RGCs and green fluorescent microspheres restricted within the tadpole eye. Scale bar, 200 m. The single-plane, high-power view of a stage 45 retina reveals the morphology of a rhodamine– dextran-labeled RGC surrounded by green fluorescent microspheres. Scale bar, 5 m. C, Effects of altered retinal neurotrophins on RGC axonal arborization in the tectum. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 43 tadpole retina, and the morphology of DiI- or YFP-labeled RGC axon arbors was visualized 24 and 48 hr later. Confocal microscope images of a control RGC axon at 0 and 24 hr demonstrate normal RGC axon arborization dynamics. Scale bar, 20 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: In Vivo, Expressing, Control, Injection, Labeling, Microscopy

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 2. Tectal BDNF retrogradely enhances RGC dendritic arborization. To determine whether tectal BDNF influences RGC dendritic arborization within the retina, tadpoles received tectal injections of microspheres treated with control, BDNF, or anti-BDNF function-blocking antibodies. Microsphere-containing neurons colabeled with rhodamine–dextran were analyzed morphologically (Fig. 1A). A, Image reconstructions of two rhodamine-labeled RGCs with simple and complex dendritic arbors illustrate differences in dendritic arbor morphologies. B, Images of RGC dendritic arbors reveal that increasing tectal BDNF enhances RGC dendritic arborization, whereas neutralizing endogenous tectal BDNF with function-blocking antibodies reduces RGC dendritic arborization. C, Quantitative analysis reveals that primary dendrite number, branch tip number, branch tips per primary dendrite, and overall dendritic length were significantly enhanced by increasing tectal BDNF and reduced by injecting anti-BDNF into the optic tectum. Scale bar, 5 m. Error bars indicate SEM. p 0.05; p 0.01; p 0.001.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Blocking Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 4. Retinal BDNF inhibits RGC den- dritic arborization in a dose-dependent man- ner. To determine whether RGCs are sensi- tive to the concentration of BDNF in the retina, Xenopus retinas were microinjected with 1–100 ng/l BDNF or control micro- spheres at the onset of dendritic arborization. Quantitative measures of dendritic arbor morphology revealed a dose-dependent re- sponse to BDNF. The highest concentration of BDNF most dramatically decreased pri- mary dendrite number, branch tip number, tips per dendrite, and dendrite length versus control. Error bars indicate SEM. p 0.05; p 0.01; p 0.001.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 5. RGC dendritic arborization is temporally sensitive to increased reti- nal BDNF levels. To determine whether RGCs were sensitive to enhanced retinal BDNF in a stage-dependent manner, control or BDNF-treated microspheres were injected into Xenopus retinas at stage 38 or 42. A, The morphology of RGC dendritic arbors revealed a stage- dependent response to increased retinal BDNF levels. B–C, Quantitative analy- sis of dendritic differentiation indicates that earlier exposure to exogenous BDNF (stages 38–45) inhibited den- dritic arborization more dramatically than later exposure to BDNF (stages 42–45). Primary dendrite number as well as dendritic branching was signifi- cantly decreased by altering retinal BDNF starting at stage 38 (B), whereas altering retinal BDNF levels from stage 42 onward (C) selectively reduced den- dritic branching without affecting pri- mary dendrite number. Error bars indi- cate SEM. p 0.05; p 0.001. Scale bar, 10 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Injection

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 6. RGC axon arbor complexity is unaffected by retinal BDNF levels. To deter- mine whether retinal BDNF influences RGC axon arborization at a distance, tadpoles were intraocularly injected with control, BDNF-, or anti-BDNF-treated microspheres, and the re- sulting changes in RGC axon arbor dynamics were compared with tectally applied BDNF (Cohen-Cory and Fraser, 1995; Lom and Co- hen-Cory, 1999). A, Individual RGC axon ar- bor morphologies of control, retinal BDNF, and tectal BDNF at 0 and 24 hr after treat- ment demonstrate that only tectally applied BDNF significantly alters RGC axon ar- borization. B, C, Altering retinal BDNF lev- els had no significant effects on RGC axon arbor complexity as measured by the in- crease in total branch number (B) and total arbor length (C) 24 and 48 hr after treatment ( p 0.05). Error bars indicate SEM. Scale bar, 20 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Injection, Control

Journal: Journal of Neuroinflammation

Article Title: Regulatory T cells protect against brain damage by alleviating inflammatory response in neuromyelitis optica spectrum disorder

doi: 10.1186/s12974-021-02266-0

Figure Lengend Snippet: Tregs changed macrophages/microglia inflammatory status in NMOSD lesion. A Representative images of Iba1 + and Foxp3-EGFP + cells in Foxp3-EGFP/cre mice with NMOSD. Scale bar, 80 μm. B Representative images of Iba1, CD16/32, iNOS, CD206, and Arg-1 stating 7 days post-NMOSD induction. Scale bar, 20 μm. C Percentages of CD16/32/iNOS/CD206/Arg-1-positive cells verse total Iba1-positive cells. n = 5 mice for each group. ** p < 0.01, # p < 0.0001. ns, no significance, versus NMOSD group; statistical analyses were performed by one-way analysis of variance followed by Dunnett’s post hoc test. D Representative western blots of CD16/32/iNOS/CD206/Arg-1 in lesion samples. E Quantitative analysis of CD16, iNOS, CD206, and Arg-1 relative expression. n = 4–6 mice for each group. * p < 0.05, ** p < 0.01. ns, no significance, versus NMOSD group; statistical analyses were performed by one-way analysis of variance followed by Dunnett’s post hoc test

Article Snippet: Frozen sections were permeabilized with Immunostaining Permeabilization Solution with Triton X-100 (Beyotime, P0096), blocked with QuickBlockTM Blocking Buffer for Immunol Staining (Beyotime, P0260), and then immunostained with the following primary antibodies at 4 °C overnight: rabbit polyclonal anti-AQP4 (1:50, Proteintech), mouse anti-glial fibrillary acidic protein (GFAP) (1:200, Cell Signaling Technology), rat anti-myelin basic protein (MBP) (1:50, Millipore), rabbit polyclonal anti-ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Wako), rabbit anti-CD45 (1:200, Servicebio), rabbit polyclonal anti-lymphocyte antigen 6G (Ly-6G) (1:200, Servicebio), rat anti-CD3 (1:200, Bio-rad), rat anti-CD68 (1:500, Bio-rad), rat anti-CD16 (1:50, BD Biosciences), goat anti-CD206 (1:100, R&D systems), rabbit anti-arginase-1 (Arg-1) (1:200, Santa Cruz Biotechnology), and rabbit anti-inducible Nitric-Oxide Synthase (iNOS) (1:200, Proteintech) followed by the appropriate Alexa Fluor conjugated secondary antibody (1:400, Invitrogen) for 1 h at room temperature.

Techniques: Western Blot, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes

doi: 10.3389/fnmol.2017.00408

Figure Lengend Snippet: Expression patterns of Kir4.1 channels and BDNF in cultured astrocytes. (A) Representative images of double-immunofluorescent staining for GFAP and Kir4.1 (a) , GFAP and BDNF (b) , or Kir4.1 and BDNF (c) in single astrocytes. (B) Clustered astrocytes stained for GFAP and BDNF in culture preparations. Lower panels showed magnified images of region indicated by squares (a–d) . Scale bar: 50 μm ( Aa , Ab , and B ) or 25 μm (Ac) .

Article Snippet: Astrocytes plated on glass coverslips were fixed in 4% paraformaldehyde, and then blocked in 0.3% Triton X-100 in phosphate buffered saline containing 1% bovine serum albumin (BSA; Wako, Osaka, Japan) for 30 min. After blocking, astrocytes were incubated with primary antibodies for glial fibrillary acidic protein (GFAP: a specific marker for astrocytes) (mouse monoclonal, 1:200; Progen, Heidelberg, Germany, RRID: AB_1541328 ), Kir4.1 (rabbit polyclonal, 1:200; Alomone Labs, Jerusalem, Israel, RRID: AB_2040120 ), BDNF (goat polyclonal, 1:200; Santa Cruz Biotechnology, Dallas, TX, United States, RRID: AB_2227954 ), and neuronal nuclear antigen (NeuN: a specific marker for neurons) (mouse monoclonal, 1:500; Millipore, Billerica, MA, United States, RRID: AB_2298772 ) at 4°C overnight.

Techniques: Expressing, Cell Culture, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes

doi: 10.3389/fnmol.2017.00408

Figure Lengend Snippet: Relationship between potencies of antidepressants for the blockade of Kir4.1 channels and for the astrocytic BDNF induction. (A) Inhibition percentage (I Drug /I Control ) of Kir4.1-conducted currents by sertraline (30 μM), fluoxetine (30 μM), imipramine (100 μM), fluvoxamine (100 μM), or mianserin (100 μM) in HEK293T cells. (B) Enhancement of BDNF mRNA expression by sertraline, fluoxetine, imipramine, fluvoxamine, or mianserin in astrocytes. Astrocytes were treated with each antidepressant (30 μM) for 8 h and the BDNF mRNA expression was analyzed by RT-PCR. The BDNF mRNA levels are expressed as the ratio to GAPDH mRNA. Each point represents the mean ± SEM of six separate experiments. ∗ P < 0.05, significantly different from the astrocytes treated with vehicle.

Article Snippet: Astrocytes plated on glass coverslips were fixed in 4% paraformaldehyde, and then blocked in 0.3% Triton X-100 in phosphate buffered saline containing 1% bovine serum albumin (BSA; Wako, Osaka, Japan) for 30 min. After blocking, astrocytes were incubated with primary antibodies for glial fibrillary acidic protein (GFAP: a specific marker for astrocytes) (mouse monoclonal, 1:200; Progen, Heidelberg, Germany, RRID: AB_1541328 ), Kir4.1 (rabbit polyclonal, 1:200; Alomone Labs, Jerusalem, Israel, RRID: AB_2040120 ), BDNF (goat polyclonal, 1:200; Santa Cruz Biotechnology, Dallas, TX, United States, RRID: AB_2227954 ), and neuronal nuclear antigen (NeuN: a specific marker for neurons) (mouse monoclonal, 1:500; Millipore, Billerica, MA, United States, RRID: AB_2298772 ) at 4°C overnight.

Techniques: Inhibition, Control, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes

doi: 10.3389/fnmol.2017.00408

Figure Lengend Snippet: Effects of Kir4.1 knockdown on BDNF and other neurotrophic factors in astrocytes. Effects of Kir4.1 siRNA transfection on BDNF mRNA (A) and protein levels (B) . The BDNF mRNA levels and protein levels were analyzed by RT-PCR and ELISA, respectively, in astrocytes at 24 or 48 h after the transfection of Kir4.1 siRNA. (C) Effects of Kir4.1 siRNA transfection on mRNA levels of GDNF, CNTF, and NGF. The GDNF, CNTF, or NGF mRNA level was analyzed by RT-PCR in astrocytes at 24 or 48 h after transfection of Kir4.1 siRNA. The mRNA and protein levels are expressed as the ratio to GAPDH mRNA and β-actin protein, respectively. Each point represents the mean ± SEM of 5–6 separate experiments. ∗ P < 0.05, ∗∗ P < 0.01, significantly different from the negative control.

Article Snippet: Astrocytes plated on glass coverslips were fixed in 4% paraformaldehyde, and then blocked in 0.3% Triton X-100 in phosphate buffered saline containing 1% bovine serum albumin (BSA; Wako, Osaka, Japan) for 30 min. After blocking, astrocytes were incubated with primary antibodies for glial fibrillary acidic protein (GFAP: a specific marker for astrocytes) (mouse monoclonal, 1:200; Progen, Heidelberg, Germany, RRID: AB_1541328 ), Kir4.1 (rabbit polyclonal, 1:200; Alomone Labs, Jerusalem, Israel, RRID: AB_2040120 ), BDNF (goat polyclonal, 1:200; Santa Cruz Biotechnology, Dallas, TX, United States, RRID: AB_2227954 ), and neuronal nuclear antigen (NeuN: a specific marker for neurons) (mouse monoclonal, 1:500; Millipore, Billerica, MA, United States, RRID: AB_2298772 ) at 4°C overnight.

Techniques: Knockdown, Transfection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes

doi: 10.3389/fnmol.2017.00408

Figure Lengend Snippet: Effects of U0126, SB202190, and SP600125 on BDNF mRNA induction by Kir4.1 knockdown in astrocytes. Astrocytes were pretreated with 10 μM U0126, 10 μM SB202190, or 10 μM SP600125 for 30 min, and transfected with Kir4.1 siRNA or a negative control. The mRNA expression of Kir4.1 (A) or BDNF (B) was analyzed by RT-PCR in astrocytes at 24 h after transfection of Kir4.1 siRNA. The mRNA levels are expressed as the ratio to GAPDH mRNA. Each point represents the mean ± SEM of 5–6 separate experiments. ∗ P < 0.05, ∗∗ P < 0.01, significantly different from the negative control.

Article Snippet: Astrocytes plated on glass coverslips were fixed in 4% paraformaldehyde, and then blocked in 0.3% Triton X-100 in phosphate buffered saline containing 1% bovine serum albumin (BSA; Wako, Osaka, Japan) for 30 min. After blocking, astrocytes were incubated with primary antibodies for glial fibrillary acidic protein (GFAP: a specific marker for astrocytes) (mouse monoclonal, 1:200; Progen, Heidelberg, Germany, RRID: AB_1541328 ), Kir4.1 (rabbit polyclonal, 1:200; Alomone Labs, Jerusalem, Israel, RRID: AB_2040120 ), BDNF (goat polyclonal, 1:200; Santa Cruz Biotechnology, Dallas, TX, United States, RRID: AB_2227954 ), and neuronal nuclear antigen (NeuN: a specific marker for neurons) (mouse monoclonal, 1:500; Millipore, Billerica, MA, United States, RRID: AB_2298772 ) at 4°C overnight.

Techniques: Knockdown, Transfection, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: Inhibition of Inwardly Rectifying Potassium (Kir) 4.1 Channels Facilitates Brain-Derived Neurotrophic Factor (BDNF) Expression in Astrocytes

doi: 10.3389/fnmol.2017.00408

Figure Lengend Snippet: Schematic drawing illustrating the effects of the Kir4.1 inhibition on the BDNF expression in astrocytes. Inhibition (blockade and knockdown) of Kir4.1 channels activates the Ras/Raf/MEK/ERK signaling pathway and enhances BDNF expression in astrocytes, which modulates the development of epilepsy (epileptogenesis) and other neuropsychiatric disorders (e.g., major depression).

Article Snippet: Astrocytes plated on glass coverslips were fixed in 4% paraformaldehyde, and then blocked in 0.3% Triton X-100 in phosphate buffered saline containing 1% bovine serum albumin (BSA; Wako, Osaka, Japan) for 30 min. After blocking, astrocytes were incubated with primary antibodies for glial fibrillary acidic protein (GFAP: a specific marker for astrocytes) (mouse monoclonal, 1:200; Progen, Heidelberg, Germany, RRID: AB_1541328 ), Kir4.1 (rabbit polyclonal, 1:200; Alomone Labs, Jerusalem, Israel, RRID: AB_2040120 ), BDNF (goat polyclonal, 1:200; Santa Cruz Biotechnology, Dallas, TX, United States, RRID: AB_2227954 ), and neuronal nuclear antigen (NeuN: a specific marker for neurons) (mouse monoclonal, 1:500; Millipore, Billerica, MA, United States, RRID: AB_2298772 ) at 4°C overnight.

Techniques: Inhibition, Expressing, Knockdown

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 1. Endogenous Rheb and GFP–Rheb are localized on the Golgi. (A) HEK293T cells were cultured in DMEM for 24 h and then fixed and stained for immunofluorescence with anti-Rheb antibody (1:200) and anti-GM130 (1:3200) diluted in blocking buffer. For Rheb knockdown, HEK293T cells were transfected with control siRNA (scramble) or Rheb siRNA. After 48 h of transfection, cells were fixed and stained with the indicated antibodies. The right row shows higher magnification views of the boxed area. (B) HEK293T cells were transiently transfected with plasmid pMRX-puro harboring GFP–Rheb for 24 h and then fixed and stained with anti-GM130 antibody (1:3200). The right row shows higher magnification views of the boxed area. (C) HEK293T cells were transiently transfected with GFP–Rheb for 24 h and then starved for 1 h in amino acid-free DMEM followed by re-stimulation using DMEM supplemented with dialyzed FBS for 10 min. The cells were fixed and stained with anti-mTOR antibody (1:400) and anti- Lamp1 antibody (1:1000). Scale bars: 5 μm.

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Cell Culture, Staining, Immunofluorescence, Blocking Assay, Knockdown, Transfection, Control, Plasmid Preparation

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 2. Stable expression of Rheb can activate mTORC1. (A) MEF cells with or without stable GFP or GFP–Rheb expression were starved in amino acid- free DMEM for 60 min and then were lysed and immunoblotted with anti- phosphorylated S6K (P-S6K) (1:1000), anti-S6K (1:1000), anti-4E-BP1 (1:1000), anti-α-tubulin (1:10,000) and anti-GFP (1:1000) antibodies. The ratio of P-S6K to S6K was quantified from three independent experiments. Bars represent the means±s.e.m. ***P<0.001 (two-tailed paired Student’s t-test). (B) LAMTOR1-knockout (p18KO) MEF cells and wild-type (WT) MEF cells were infected for 48 h with the viruses transducing the indicated proteins and starved for amino acids as described in A. The cells were then lysed and immunoblotted with the indicated antibodies, as above. The ratio of P-S6K to S6K was quantified from three independent experiments. Bars represent the means±s.e.m. ***P<0.001 (two-tailed paired Student’s t-test).

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Expressing, Two Tailed Test, Knock-Out, Infection

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 3. Golgi-localized Rheb can activate mTORC1. (A) A schematic of the method of targeting of GFP–Rheb to the Golgi with FLAG–GBP-tagged Rab1A. (B) MEF cells stably expressing GFP–Rheb with FLAG–GBP–Rab1A or FLAG–GBP–Rab7 were fixed and stained with anti-GM130 (1:1000) or anti-Lamp1 (1:1000) antibodies. For GFP–Rheb and FLAG–GBP–Cytb co-expression, the cells were transfected with the RFP–Sec61β ER marker and then fixed and observed. The right row shows higher magnification views of the boxed area. Scale bars: 5 μm. (C) Colocalization quantified as Pearson’s correlation coefficient (PCC) between GFP–Rheb and GM130, Lamp1 or RFP–Sec61β. The localization of GFP–Rheb with Lamp1 or RFP–Sec61β in FLAG–GBP–Rab1A-expressing cells, GFP–Rheb with GM130 or RFP–Sec61β in FLAG-GBP-Rab7-expressing cells, GFP–Rheb with GM130 or Lamp1 in FLAG–GBP–Cytb-expressing cells is shown in Fig. S2. More than 30 cells were quantified. Bars represent the means±s.e.m. (D) MEF cells stably expressing GFP, GFP–Rheb, or GFP–Rheb with FLAG–GBP, FLAG–GBP–Rab1A, FLAG–GBP–Rab33B, FLAG–GBP–Rab7, FLAG–GBP–Rab9A or FLAG–GBP–Cytb were starved for 60 min in amino acid-free DMEM. Cells were then lysed and immunoblotted with anti-FLAG antibody (1:1000) and the indicated antibodies as in Fig. 2. (E) Quantification of the ratio of P-S6 K to S6 K from blots as shown D (three independent experiments). Bars represent the means±s.e.m. *P<0.05 (two-tailed paired Student’s t-test).

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Stable Transfection, Expressing, Staining, Transfection, Marker, Two Tailed Test

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 5. The Golgi contacts the lysosome at the perinuclear region. (A) Duolink, using anti-GM130 (1:000) and anti-Lamp1 (1:000) antibodies, was used to detect the GLCS in MEF cells stably expressing GFP–Rheb. The right row shows higher magnification views of the boxed area. (B) The cells described in Awere treated with 1 μg/ml BFA for 2 h and then fixed and subjected to Duolink for detection of the GLCS. The right row shows higher magnification views of the boxed area. (C) MEF cells stably expressing GFP–Gal3 were treated without or with 1000 μM LLOMe for 3 h. After washing LLOMe out for 2 h in DMEM, the cells were subjected to Duolink for detection of the GLCS. (D) Quantification of the number of GLCSs from three independent experiments as shown in C. Bars represent the means±s.e.m. ***P<0.001, two-tailed paired Student’s t-test. Scale bars: 5 μm.

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Stable Transfection, Expressing, Two Tailed Test

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 6. Golgi or lysosomal rupture suppresses mTORC1 activity and GLCS number increases in response to amino acids. (A) MEF cells stably expressing GFP–TFEB were treated with 1 μg/ml of BFA for 2 h and then fixed and incubated with anti-GM130 antibody (1:1000). Scale bars: 5 μm. (B) Quantification of the percentage of nuclear-localized GFP–TFEB from three independent experiments as shown in A. Bars represent the means±s.e.m. ***P<0.001 (two-tailed paired Student’s t-test). (C) GFP–TFEB was transiently expressed in MEF cells stably expressing mSt–Gal3. The cells were treated without or with 1000 μM LLOMe for 3 h. After washing for 2 h in DMEM, the cells were fixed and observed. Scale bars: 5 μm. (D) Quantification of the percentage of nuclear-localized GFP–TFEB from three independent experiments as shown in C. Bars represent the means±s.e.m. ***P<0.001 (two-tailed paired Student’s t-test). (E) MEF cells were cultured in DMEM (Nutrient), starved for 60 min in amino acid- free DMEM (S: 1 h), or replenished for 5 or 10 min with amino acids in DMEM (S: 1 h +AA 5min or 10min, respectively). Cells were then fixed and stained with DAPI (1:1000) and anti-Lamp1 antibody (1:1000). Scale bars: 10 μm. (F) Duolink, using anti-GM130 antibody (1:1000) and anti-Lamp1 antibody (1:1000), was used to detect the GLCS in MEF cells stably expressing GFP–Rheb after being starved for 60 min in amino acid- free DMEM or replenished for 10 min with amino acids in DMEM. Scale bars: 25 μm. (G) The number of GLCS per cell was counted in more than 100 cells treated as in E. Bars represent the means±s.e.m. from three independent experiments. *P<0.05 (two-tailed paired Student’s t-test).

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Activity Assay, Stable Transfection, Expressing, Incubation, Two Tailed Test, Cell Culture, Staining

Journal: Journal of cell science

Article Title: Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi-lysosome contact site.

doi: 10.1242/jcs.208017

Figure Lengend Snippet: Fig. 7. Proposed model – Rheb activates mTORC1 at the GLCS. Rheb at least partially localizes to the Golgi. When the amino acids signal to the cells, mTORC1 translocates to lysosomes. To respond to the amino acid signals, the lysosomes accumulate at the perinuclear region, where the Golgi exists, leading to the formation of more GLCSs to provide a platform for the activation of mTORC1 by Rheb.

Article Snippet: Wild-type Rheb, cloned from mouse cDNA, Raptor, cloned from Myc-Raptor (Hao et al., 2016), and TSC2, cloned from pcDNA3 Flag TSC2 obtained from Addgene (#14129, Cambridge, MA) (Manning et al., 2002), were inserted into the BamHI and NotI sites of pMRX-puro-EGFP (Itoh et al., 2011).

Techniques: Activation Assay