Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BP1 tethers the TSC complex to lysosomes (A) PLA of TSC2-LAMP2 in insulin/aa-stimulated siG3BP1 cells (15 min, 1 μM insulin). PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 100 μm. n = 4. (B) Quantitation of data in (A). Shown are data points and mean ± SEM. Control (0 min) normalized to 1. n = 12 technical replicates. (C) IF of LAMP1-TSC2 co-localization in G3BP1 KO cells transfected with siRHEB; insulin/aa stimulation (1 μM insulin). Overlay: white, LAMP1-TSC2 co-localization; green, TSC2; magenta, LAMP1; insert, magnification of the yellow square. Scale bar, 10 μm. n = 3. (D) Quantitation of data in (C). Shown are data points and mean ± SEM. (E) TSC2 KO cells in full medium. n = 3. (F) Quantitation of TSC2 in (E). Shown are data points and mean ± SEM. (G) Quantitation of RPS6KB1-pT389 in (E). Data are shown as in (F). (H) Size of G3BP1 KO cells. Mean ± SEM. *p < 0.05. n = 3. (I) Quantitation of data in (J). Shown are data points and mean ± SEM. (J) IF of MTOR-LAMP2 co-localization in G3BP1 KO cells. Overlay: white, MTOR-LAMP2 co-localization; green, MTOR; magenta, LAMP2; insert: magnification of the yellow square. Scale bar, 10 μm. n = 3. (K) Insulin-stimulated TSC2 KO cells transfected with siG3BP1. n = 4. (L) Quantitation of TSC2 in (K). Shown are data points and mean ± SEM. TSC2 was compared between control and TSC2 KO cells. (M) Quantitation of G3BP1 in (K). Shown are data points and mean ± SEM. G3BP1 was compared between siControl and siG3BP1 in control or TSC2 KO cells. (N) Quantitation of RPS6KB1-pT389 in (K). Data are shown as in (M). See also Figure S4 .

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Quantitation Assay, Control, Transfection

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BP1 and G3BP2 reside at lysosomes (A) Quantitation of data in (B). G3BP1, green area. Mean ± SEM. (B) Sucrose density gradient separation of serum/aa-starved MCF-7 cells. n = 3. (C) Lyso-prep with ferromagnetic nanoparticles. PNS, postnuclear supernatant. n = 3. (D) PLA of G3BP1-LAMP1 in serum/aa-starved G3BP1 KO cells. PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 10 μm. n = 3. (E) Quantitation of data in (D). Shown are data points and mean ± SEM. n = 8 technical replicates. (F) Trypsin digest of lyso-preps prepared as in (C). n = 3 except for TSC2 (n = 2). (G) IP against TSC2 (TSC2 #1) or mock (mouse IgG). n = 3. (H) PLA of G3BP1-TSC2 in serum/aa-starved G3BP1 KO cells. PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 10 μm. n = 4. (I) Quantitation of data in (H). Shown are data points and mean ± SEM. n = 8 technical replicates. (J) IP against MTOR or mock (rat IgG); insulin/aa-stimulated shG3BP1 #1 cells (15 min). n = 4. (K) Quantitation of G3BP1 in (J). Shown are data points and mean ± SEM. (L) Quantitation of TSC2 in (J). Data are shown as in (K). (M) IP against TSC2 (TSC2 #2 or #3) or mock (rabbit IgG). n = 3. (N) IP against TSC2 (TSC2 #2) or mock (rabbit IgG); insulin/aa-stimulated shG3BP1 #1 cells (15 min). n = 4. (O) Quantitation of TSC1 in (N). Shown are data points and mean ± SEM. (P) Quantitation of G3BP1 in (N). Data are shown as in (O). (Q) Quantitation of LAMP1 in (N). Data are shown as in (O). See also Figure S4 .

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Quantitation Assay

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BP1 phenocopies lysosomal TSC functions, related to and (A) PLA of G3BP1-TSC2 in serum/aa-starved siG3BP1 cells. PLA puncta, white dots; nuclei, blue (DAPI). Scale bar, 10 μm. n = 3. (B) Quantitation of data in (A). Shown are data points and mean ± SEM. n = 5 technical replicates. (C) Sucrose density gradient of serum/aa-starved or insulin/aa-stimulated MCF-7 cells. n = 3. (D) Quantitation of data in (C). Area under the curve highlighted in green (G3BP1), orange (TSC2), and gray (LAMP2), starved condition; dashed lines, stimulated condition. Mean ± SEM. (E) Insulin/aa-stimulated shG3BP1 #1 cells. n = 5. (F) Quantitation of TSC2-pT1462 in (E). Shown are data points and mean ± SEM. (G) Quantitation of AKT1-pS473 in (E). Data shown as in (F). (H) Insulin/aa-stimulated siTSC2 cells. n = 6. (I) Quantitation of TSC2 in (H). Shown are data points and mean ± SEM. (J) Quantitation of RPS6KB1-pT389 in (H). Data shown as in (I). (K) Cell size of TSC2 KO cells. Mean ± SEM. *p < 0.05. n = 3. (L) IF of LAMP2 positioning in serum/aa-starved siG3BP1 cells. White, LAMP2; blue (Hoechst), nuclei. Scale bar, 10 μm. n = 3.

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Quantitation Assay

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BPs bridge TSC2 to LAMP proteins (A) IP against TSC1 (TSC1 #1) or mock (rabbit IgG) in TSC2 KO cells. n = 3. (B) IP against GFP or FLAG; transfection with the indicated plasmids. n = 5. (C) IP against TSC1 (TSC1 #2) or mock (mouse IgG) incubated with NaCl or SDS. n = 3. (D) IP against GFP or mock (mouse IgG); transfection with the indicated plasmids. n = 3. (E) BiFC. Protein+C-terminal mLumin is indicated first; protein+N-terminal mLumin is indicated second. TL, transmitted light. Scale bar, 100 μm. n = 3. (F) Quantitation of data in (E). Shown are data points and mean ± SEM. (G) IP against MTOR or mock (rat IgG). n = 3. (H) BiFC. Protein+C-terminal mLumin is indicated first; protein+N-terminal mLumin is indicated second. TL, transmitted light. Scale bar, 100 μm. n = 4. (I) Quantitation of data in (H). Shown are data points and mean ± SEM. (J) Quantitation of data in (K). Shown are data points and mean ± SEM. (K) BiFC. Protein+C-terminal mLumin is indicated first; protein+N-terminal mLumin is indicated second. TL, transmitted light. Scale bar, 100 μm. n = 5. (L) BiFC. Protein+C-terminal mLumin is indicated first; protein+N-terminal mLumin is indicated second. TL, transmitted light. Scale bar, 100 μm. n = 3. (M) Quantitation of data in (L). Shown are data points and mean ± SEM. (N) IP against FLAG or mock (mouse IgG); transfection with the indicated plasmids. n = 3. (O) IP against FLAG or mock (mouse IgG); transfection with the indicated plasmids. n = 3. (P) Phylogenetic analysis. Black square, protein present in species. See also Figure S5 .

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Transfection, Incubation, Quantitation Assay

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BP1 suppresses mTORC1-driven migration in breast cancer cells (A) Scratch assay with shG3BP1 #1 cells. Scale bar, 150 μm. n = 3. (B) Quantitation of data in (A). Shown are data points and mean ± SEM. (C) Real-time cell analysis (RTCA) of proliferation of shG3BP1 #1 MCF-7 cells. Mean ± SEM. n = 6. (D) Quantitation of data in (C). Shown are data points and mean ± SEM. (E) Transwell migration of G3BP1 KO cells (6–8 h). Scale bar, 150 μm. n = 5. (F) Quantitation of data in (E). Shown are data points and mean ± SEM. (G) G3BP1 mRNA expression. Expression values from The Cancer Genome Atlas (TCGA) processed and normalized by RNA-Seq by Expectation Maximization (RSEM) are classified according to PAM50. Data are shown as boxplots, median with 25 th +75 th percentiles as boxes, and 5 th +95 th percentiles as whiskers. (H) Relapse free survival (RFS) of individuals with breast cancer based on G3BP1 RNA levels. (I) RFS of individuals with breast cancer based on G3BP1 protein levels. (J) RFS of individuals with breast cancer based on TSC1 RNA levels. (K) RFS of individuals with breast cancer based on TSC2 RNA levels.

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Migration, Wound Healing Assay, Quantitation Assay, Cell Analysis, Expressing, RNA Sequencing

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet: G3BP1 deficiency elicits mTORC1-driven neuronal phenotypes in vivo (A) IP against TSC1 (TSC1 #3) or mock (rabbit IgG). n = 2. (B) Zebrafish larvae injected with g3bp1 MO. dpf, days post fertilization. n = 4/day. (C) Quantitation of Rps6-pS235/236 in (B), pooled for 2+3 dpf. Shown are data points and mean ± SEM. (D) Dorsal and lateral view of a zebrafish larva brain. P, pallium; OT, optic tectum; H, habenula; Cb, cerebellum; OB, olfactory bulb; SP, subpallium; Th, thalamus; Tub, tuberculum; T, tegmentum; HTh, hypothalamus. (E) IF of Rps6-pS235/236 in g3bp1 MO-injected zebrafish larvae. Nuclei, blue (DAPI); dashed white lines, white matter (WM) compartments of the pallium; arrows, Rps6-pS235/236-positive cells in the WM. Scale bar, 25 μm. n ≥ 29 larvae/condition. (F) Quantitation of Rps6-pS235/236-positive cells in the pallium in (E). Shown are data points and mean ± SEM. (G) Quantitation of cells in the WM in (E). Data are shown as in (F). (H) Quantitation of Rps6-pS235/236-positive cells in the WM in (E). Data are shown as in (F). (I) Quantitation of HuC-positive cells in g3bp1 MO zebrafish larvae (24 hpf [hours post fertilization]). Shown are data points and mean ± SEM. n ≥ 10 larvae/condition. (J) Movement speed of single HuC-positive cells. Data are shown as in (I). (K) Track duration of single HuC-positive cells. Data are shown as in (I). Arrow, maximum track duration. (L) Quantitation of epileptiform events in LFP recordings from the pallia of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 34 larvae/condition. (M) Representative LFP recordings for (L). (N) Quantitation of epileptiform events in LFP recordings from optic tecta of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 20 larvae/condition. (O) Representative LFP recordings for (N). (P) Neuronal activity in pallia of Tg ( HuC:GCaMP5G ) zebrafish larvae injected with g3bp1 MO (4 dpf). Dashed white lines, pallium; arrows, ectopic cells with high neuronal activity in the WM; yellow/orange, high neuronal activity. Scale bar, 25 μm. n ≥ 27 larvae/condition. (Q) Quantitation of active neuronal cells in (P). Shown are data points and mean ± SEM. (R) Quantitation of mean neuronal activity in the subpallia of Tg ( HuC:GCaMP5G ) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 15 larvae/condition. (S) Quantitation of mean neuronal activity in the WM of Tg ( HuC:GCaMP5G ) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 14 larvae/condition. (T) Quantitation of rapamycin-mediated fold reduction in the activity of single cells in the WM of Tg ( HuC:GCaMP5G ) zebrafish larvae injected with g3bp1 MO (4 dpf). The number of active cells in rapamycin-treated larvae was normalized to those in untreated larvae. Shown are data points and mean ± SEM. n ≥ 14 larvae/condition. (U) Quantitation of GABAergic cells in optic tecta of Tg ( dlx5a/dlx6a-EGFP ) x Tg ( vglut2a:loxP-RFP-loxP-GFP ) zebrafish larvae injected with g3bp1 MO (4 dpf). Shown are data points and mean ± SEM. n ≥ 34 larvae/condition. (V) Quantitation of glutamatergic cells in optic tecta of Tg ( dlx5a/dlx6a-EGFP ) x Tg ( vglut2a:loxP-RFP-loxP-GFP ) zebrafish larvae injected with g3bp1 MO (4 dpf). Data are shown as in (U). (W) Locomotor activity of tsc2 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 26 larvae/condition. (X) Locomotor activity of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n ≥ 36 larvae/condition. (Y) Locomotor activity of g3bp1 MO zebrafish larvae (4 dpf). Mean ± SEM. n = 24, untreated, n = 36 ethosuximide-treated larvae/condition. See also Figure S6 , , and and .

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: In Vivo, Injection, Quantitation Assay, Activity Assay

Journal: Cell

Article Title: G3BPs tether the TSC complex to lysosomes and suppress mTORC1 signaling

doi: 10.1016/j.cell.2020.12.024

Figure Lengend Snippet:

Article Snippet: TSC2 , Cell Signaling , Cat# 4308; RRID: AB_10547134.

Techniques: Virus, Recombinant, Protease Inhibitor, Modification, In Situ, Immunoprecipitation, Saline, Membrane, Affinity Purification, Transfection, Blocking Assay, Bicinchoninic Acid Protein Assay, Western Blot, Lysis, shRNA, RNA Expression, Expressing, Control, Software, Sequencing, Cell Culture, Microscopy, Electrophoresis, Imaging

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pcDNA3 Flag TSC2 (plasmid) , Addgene , RRID: Addgene_14129 , .

Techniques: Transfection, Construct, shRNA, Transduction, Knock-Out, Recombinant, Plasmid Preparation, Sequencing, Control, cDNA Synthesis, Software

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Phospho- TSC2-S939 (rabbit polyclonal) , Cell Signaling Technology , Cat# 3615 , WB (1:1000).

Techniques: Transfection, Construct, shRNA, Transduction, Knock-Out, Recombinant, Plasmid Preparation, Sequencing, Control, cDNA Synthesis, Software

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 1 TSC2 gene suppression activates mTORC1 signalling during nutrient deprivation and hypoxia. (A) LNT-229 and LN-308 TSC2sh and control cells (non-targeting sequence, NTsh) were analysed by qPCR. TSC2 gene suppression was confirmed. Values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (B) LNT-229 and LN-308 cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-Akt (Ser 473), Akt, P-S6K1 (Thr 389), S6K1, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. The same incubation conditions were used for immunoprecipitation of GTP-RHEB. The precipitate was then analysed by immunoblot with an antibody for RHEB as well as an immunoblot with an antibody for actin as a loading control of the employed lysate (lower two lanes). (C) G55 (left) and LN-428 (right) NTsh and TSC2sh cells were analysed by qPCR. TSC2 gene suppression was confirmed, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD). (D) G55 (left) and LN-428 (right) cells were incubated for 4 h, as indicated. Cellular lysates were analysed by immunoblot with antibodies for TSC2, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. (E) LNT-229 and LN-308 cells were incubated in serum-free and serum containing (10% FCS) culture conditions without glucose restriction (25 mM glucose) for 5 days. Cell density was measured by crystal violet staining at the beginning of cultivation and after 5 days (n = 4, mean SD).

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Control, Sequencing, Gene Expression, Incubation, Western Blot, Immunoprecipitation, Staining

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 2 TSC2 gene suppression sensitizes human malignant glioma cells to hypoxia-induced cell death. Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h), ATP depletion (12 h) or an increase in ROS (8 h) was observed. (A) Cell death was quantified by LDH release and by propidium iodide staining (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (B) G55 and LN-428 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen until cell death (24 h) was observed. Cell death was quantified by LDH release (n = 4, mean SD, *P 5 0.05, **P 5 0.01). (C) ATP was quantified by a luciferase-based assay. The ratio of ATP concentrations in hypoxia to normoxia is depicted (n = 5, mean SD, **P 5 0.01). (D) ROS levels were measured by H2DCFDA-FACS (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant; ROS = reactive oxygen species.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Staining, Luciferase

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 3 TSC2sh gene suppression has no effect on glucose consumption and lactate production, but alters metabolic pathways. (A) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Glucose consumption and lactate production were determined in the supernatant (n = 3, mean SD). (B) Cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 8 h. Intracellular metabolites were analysed either by LC-MS-MS analysis or GC-MS analysis (n = 3, mean SD, *P 5 0.05, **P 5 0.01). (C) LNT-229 and LN-308 NTsh and TSC2sh cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic conditions or 0.1% oxygen for 4 h, cDNA and cellular lysates of cells were generated. Gene expression of G6PD was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05) (left). Cellular lysates were analysed by immunoblot with antibodies for G6PD or actin. Quantification of G6PD protein content of three independent experiments is provided (n = 3, mean SD, *P 5 0.05, **P 5 0.01) and one representative immunoblot is shown (middle). LNT-229 and LN-308 wild-type cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin, 200 nM torin2 or vehicle for 72 h. Cellular lysates were analysed by immunoblot with antibodies for G6PD, P-S6RP (Ser 240/244 and Ser 235/235), S6RP, P-4E-BP1 (Ser 65), 4E-BP1 or actin. n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Liquid Chromatography with Mass Spectroscopy, Gas Chromatography-Mass Spectrometry, Generated, Gene Expression, Western Blot

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 4 TSC2 gene suppression induces oxygen consumption and expression of genes of mitochondrial oxidative function. (A) LNT-229 NTsh and TSC2sh cells were incubated in medium containing 10% FCS with 25 mM glucose. Oxygen consumption was measured by a fluorescence-based assay (left, n = 3, mean, **P 5 0.01). Using an Oxygraph-2k system steady state oxygen consumption was also monitored (right, n = 5 mean + SD, **P 5 0.01). (B) cDNA of LNT-229 and LN-308 NTsh and TSC2sh cells cultured in standard conditions was generated. Gene expression of PPARGC1A/PGC-1a and PPARGC1B/PGC-1b, ERRSA/ERR-a, NRF1, MT-CYB, MT-CO1, MT-CO2, SCO2, ATP5G1, COX5A and IDH3A was quantified by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, *P 5 0.05, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Expressing, Incubation, Cell Culture, Generated, Gene Expression

Journal: Brain : a journal of neurology

Article Title: Mammalian target of rapamycin complex 1 activation sensitizes human glioma cells to hypoxia-induced cell death.

doi: 10.1093/brain/awx196

Figure Lengend Snippet: Figure 6 Inhibition of mTORC1 with rapamycin reverts the phenotype of TSC2sh cells. (A) LNT-229 NTsh and TSC2sh cells were exposed to medium containing 10% FCS with 25 mM glucose with 200 nM rapamycin or vehicle. Oxygen consumption was measured with a fluorescence-based assay (n = 3, mean, *P 5 0.05, **P 5 0.01). (B) cDNA of LNT-229 NTsh and TSC2sh cells treated with 200 nM rapamycin or vehicle for 24 h in DMEM medium containing 10% FCS and 25 mM glucose was generated. Gene expression of PPARGC1A/PGC-1a, PPARGC1B/ PGC-1b and ERRSA/ERR-a was measured by qPCR, values are normalized to 18S as well as SDHA housekeeping gene expression (n = 3, mean SD, **P 5 0.01). n.s. = not significant.

Article Snippet: Generation of TSC2 gene suppressed cells The pLKO.1 plasmids targeting TSC2 (TSC2sh) and the pLKO.1 plasmid with a non-targeting shRNA sequence (NTsh) were ordered from Addgene (Addgene #15478, #1864).

Techniques: Inhibition, Generated, Gene Expression

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet: ( A ) The tandem mass spectrometry (MS/MS) datasets previously acquired by MudPIT analyses of FLAG-DYRK1A affinity purifications and negative FLAG controls were searched against the most recent releases of the human protein sequence databases (built by collating and removing redundant entries from NCBI Homo sapiens RefSeq GCF_000001405.40_GRCh38.p14 and GCF_009914755.1_T2T-CHM13v2.0 ). Highly enriched proteins include known and novel DYRK1A-interacting partners and are reported with their peptide counts and distributed normalized spectral abundance factor (dNSAF) values, which reflect their relative abundance in the samples . ( B ) Flag beads were used to pull down Flag-DYRK1A from whole cell extracts of HEK293 transfected with Flag-DYRK1A, and Protein A beads were used as a control. The blots were probed with TSC1 and TSC2 antibodies. Actin was used to normalize the lysate inputs. ( C ) Endogenous DYRK1A was immunoprecipitated with DYRK1A antibody from HEK293 cytoplasmic fraction generated using the Dignam protocol and probed with antibodies against endogenous DYRK1A, TSC1, and TSC2. Rabbit IgG was used as the IP control ( D ) Flag-DYRK1A and Flag-DYRK1A kinase domain constructs were affinity purified using Flag-beads from HEK293 cells co-transfected with HA3-TSC1 and probed with α-HA and α-Flag antibodies. Actin was used as the loading control. Figure 2—source data 1. Uncropped blots for . Figure 2—source data 2. Raw data of Flag-DYRK1A affinity purification and Flag, TSC1 and TSC2 blot for . Figure 2—source data 3. Uncropped blots for . Figure 2—source data 4. Raw data of interaction between endogenous DYRK1A and TSC1 and TSC2 for . Figure 2—source data 5. Uncropped blots for . Figure 2—source data 6. Raw data of interaction between Kinase domain of Flag-DYRK1A and HA-TSC1 for .

Article Snippet: Antibody , anti-Phospho-TSC2-T1462(rabbit polyclonal) , Abclonal , Cat# AP0866 , WB (1:1000).

Techniques: Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Transfection, Control, Immunoprecipitation, Generated, Construct, Affinity Purification

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet: Flag and HA beads were used to pull down HA3-TSC1 and Flag-TSC2 from whole cell extracts of HEK293 transfected with HA3-TSC1 and Flag-TSC2. Blots were probed with Flag, HA, and DYRK1A antibodies. Actin was used as the loading control. Figure 2—figure supplement 1—source data 1. Uncropped blots for . Figure 2—figure supplement 1—source data 2. Raw data of interaction between overexpressed TSC1 and TSC2 with endogenous DYRK1A for .

Article Snippet: Antibody , anti-Phospho-TSC2-T1462(rabbit polyclonal) , Abclonal , Cat# AP0866 , WB (1:1000).

Techniques: Transfection, Control

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet: ( A ) An in-vitro kinase assay was performed using DYRK1A and kinase-dead DYRK1A (K188R) that were purified from bacteria. Flag-TSC2 and HA3-TSC1 were co-expressed in HEK293 cells and purified using a combination of (1:1) of HA and Flag beads. Beads were equilibrated with kinase assay buffer before the reactions were initiated on beads. After incubation for 30 min at 30°C, reactions were stopped by the addition of SDS loading buffer. Since bacterially purified DYRK1A is autophosphorylated, it exhibits a fuzzier signal, whereas kinase-dead DYRK1A is incapable of phosphorylation and appears as a sharp signal. ( B, C ) RHEB overexpression partially rescues the size of HEK293 cells. HEK293 cells were first transduced with short hairpin RNA (shRNA) lentivirus targeting DYRK1A or control and selected with 1 ug/ml Puromycin for three days, after which they were re-transduced with lentivirus expressing Flag-RHEB. The concentration of Puromycin was raised to 2 ug/ml for the next 48 hr in order to select for the second round of transduction. ( B ) Panel shows knockdown efficiency of DYRK1A and overexpression of RHEB. ( C ) Lower panel shows cell size analysis. Data represent the mean ± SD (n=3 biological replicates). Student’s t-test was done to compare samples. Significant difference in p-value = *p<0.05. Figure 4—source data 1. Uncropped blots for . Figure 4—source data 2. Raw data showing in vitro kinase assay using recombinant DYRK1A and K188R for . Figure 4—source data 3. Uncropped blots for . Figure 4—source data 4. Raw data showing knockdown of DYRK1A and overexpression of Flag-RHEB for .

Article Snippet: Antibody , anti-Phospho-TSC2-T1462(rabbit polyclonal) , Abclonal , Cat# AP0866 , WB (1:1000).

Techniques: In Vitro, Kinase Assay, Purification, Bacteria, Incubation, Phospho-proteomics, Over Expression, Transduction, shRNA, Control, Expressing, Concentration Assay, Knockdown, Recombinant

Journal: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Phospho-TSC2-T1462(rabbit polyclonal) , Abclonal , Cat# AP0866 , WB (1:1000).

Techniques: Transfection, Construct, shRNA, Transduction, Knock-Out, Recombinant, Plasmid Preparation, Sequencing, Control, cDNA Synthesis, Software