Journal: bioRxiv

Article Title: TSC1 phosphorylation by lysosomal mTORC1 establishes a minimal autoregulatory feedback loop

doi: 10.64898/2026.01.15.699678

Figure Lengend Snippet: (A) Schematic representation of the mTOR-dependent phosphorylation sites on human TSC1 protein, identified by phospho-proteomics. The amino acid sequence surrounding positions T1047 and S1080 is shown, as well as the position of the N-terminal HEAT (N-HEAT), the helical linker (HL) and the coiled-coil (CC) TSC1 domains. (B) Endogenous TSC1 immunoprecipitation (IP) from TSC2 KO HEK293FT cells stably expressing wild-type human TSC2 (WT) or the N1643K GAP-inactive hTSC2 mutant (GAP mut ), followed by immunoblotting with the indicated antibodies. n = 3 independent experiments. (C) Endogenous TSC1 immunoprecipitation from MEFs treated with Torin1 (250 nM, 1 hr) or DMSO as control, followed by immunoblotting with the indicated antibodies. n = 3 independent experiments. (D) Endogenous TSC1 immunoprecipitation (IP) from HEK293FT cells treated with media containing or lacking amino acids (AAs), in basal (+), starvation (–), or add-back (–/+) conditions, followed by immunoblotting with the indicated antibodies. n = 3 independent experiments. (E) Endogenous TSC1 immunoprecipitation (IP) from MEFs treated with media containing or lacking serum, in basal (+), starvation (–), or add-back (–/+) conditions, followed by immunoblotting with the indicated antibodies. Note that S1080 in human TSC1 corresponds to S1074 in mouse TSC1. n = 3 independent experiments. (F) Endogenous TSC1 immunoprecipitation (IP) from HEK293FT cells treated with media containing or lacking glucose, in basal (+), starvation (–), or add-back (–/+) conditions, followed by immunoblotting with the indicated antibodies. n = 3 independent experiments. (G) In vitro kinase assays with endogenous mTOR immunopurified from HEK293FT cells, using recombinant His6-tagged TSC1 989-1163 as a substrate. Recombinant His 6 -tagged 4E-BP1 was used as a positive control. Substrate phosphorylation detected by immunoblotting. Addition of Torin1 (250 nM) to the tubes 10 min before the initiation of the IVK reactions was used to confirm mTOR-dependent substrate phosphorylation. n = 2 independent experiments. (H) Co-immunoprecipitation experiments with lysates from HEK293FT cells transiently expressing HA-tagged RAPTOR or a control vector reveal binding of mTORC1 to endogenous TSC1 and TSC2. The input and anti-HA IP samples were analyzed by immunoblotting with the indicated antibodies. n = 3 independent experiments. See also Figures S3 and S4.

Article Snippet: The remaining volume of the supernatants was incubated with 1 μL anti-TSC1 antibody (#6935, Cell Signaling Technology) at 4 °C in an overhead rotator for 3 h, followed by incubation with 30 μL pre-washed Protein A agarose bead slurry (#11134515001, Roche) for an additional hour at 4 °C in an overhead rotator.

Techniques: Phospho-proteomics, Sequencing, Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis, Western Blot, Control, In Vitro, Recombinant, Positive Control, Plasmid Preparation, Binding Assay

Journal: bioRxiv

Article Title: TSC1 phosphorylation by lysosomal mTORC1 establishes a minimal autoregulatory feedback loop

doi: 10.64898/2026.01.15.699678

Figure Lengend Snippet: (A) Immunoblots with lysates from TSC2 KO HEK293FT cells stably expressing wild-type human TSC2 (WT) or the N1643K GAP-inactive hTSC2 mutant (GAP mut ) and transfected with siRNAs targeting RagA/C, RagB/D, or Luciferase as a control, probed with the indicated antibodies. Torin1 (250 nM, 1 h) was used to inhibit mTOR. n = 3 independent experiments. (B) Endogenous TSC1 immunoprecipitation from TSC2 KO HEK293FT cells stably expressing the N1643K GAP-inactive hTSC2 mutant (GAP mut ) and transfected with siRNAs targeting RagA/C, Lamtor1, or Luciferase as a control, probed with the indicated antibodies. Torin1 (250 nM, 1 h) was used to inhibit mTOR. n = 3 independent experiments. (C) Endogenous TSC1 immunoprecipitation from TSC2 KO HEK293FT cells stably expressing the N1643K GAP-inactive hTSC2 mutant (GAP mut ), treated with Torin1 (250 nM, 1 h) or Bafilomycin A1 (100 nM, 8 h), probed with the indicated antibodies. n = 3 independent experiments. (D) Co-immunoprecipitation experiments with lysates from HEK293FT cells transiently expressing HA-tagged RAPTOR or a control vector and transfected with siRNAs targeting Lamtor1, or Luciferase as a control. The input and anti-HA IP samples were analyzed by immunoblotting with the indicated antibodies. n = 3 independent experiments. (E) Immunoblots with lysates from HEK293FT transiently expressing FLAG-tagged wild-type RHEB (WT) or the farnesylation-deficient RHEB Δ180 mutant (Δ180), treated with Torin1 (250 nM, 1 h) or DMSO as control, probed with the indicated antibodies. n = 2 independent experiments. (F) Schematic model illustrating the LAMTOR- and Rag-dependent phosphorylation of TSC1 on the lysosomal surface. See text for details.

Article Snippet: The remaining volume of the supernatants was incubated with 1 μL anti-TSC1 antibody (#6935, Cell Signaling Technology) at 4 °C in an overhead rotator for 3 h, followed by incubation with 30 μL pre-washed Protein A agarose bead slurry (#11134515001, Roche) for an additional hour at 4 °C in an overhead rotator.

Techniques: Western Blot, Stable Transfection, Expressing, Mutagenesis, Transfection, Luciferase, Control, Immunoprecipitation, Plasmid Preparation, Phospho-proteomics

Journal: bioRxiv

Article Title: TSC1 phosphorylation by lysosomal mTORC1 establishes a minimal autoregulatory feedback loop

doi: 10.64898/2026.01.15.699678

Figure Lengend Snippet: (A) Immunoblots with lysates from TSC1 KO HEK293FT cells transiently expressing wild-type TSC1 (WT) or the phospho-dead TSC1 2A mutant (2A), treated with MG132 (10 μM, 8 h) or DMSO as control, probed with the indicated antibodies. n = 3 independent experiments. (B) TSC1 immunoprecipitation from TSC1 KO HEK293FT cells transiently expressing wild-type TSC1 (WT) or the phospho-dead TSC1 2A mutant (2A), followed by immunoblotting with the indicated antibodies. Cells were treated with MG132 (10 μM, 8 h) to block proteasomal degradation. n = 3 independent experiments. (C-D) Immunoblots with lysates from MEFs treated with cycloheximide (CHX, 100 μM) alone or in combination with Torin1 (250 nM) for the indicated time points (C). Quantification of TSC1 protein levels in (D). n = 3 independent experiments (E-G) Immunoblots with lysates from TSC1 KO HEK293FT cells transiently expressing wild-type TSC1 (WT) or the phospho-dead TSC1 2A mutant (2A), probed with the indicated antibodies (E). Quantification of TFEB phosphorylation and S6K1 phosphorylation in (F) and (G), respectively. n = 3 independent experiments. (H-I) TFE3 localization analysis in TSC1 KO HEK293FT cells transiently expressing wild-type TSC1 (WT) or the phospho-dead TSC1 2A mutant (2A), using confocal microscopy (H). Quantification of TFE3 nuclear intensity (arbitrary units, a.u.) in (I). n = 95-108 individual nuclei. (J) Quantification of LysoTracker signal intensity (arbitrary units, a.u.) from TSC1 KO HEK293FT cells transiently expressing wild-type TSC1 (WT) or the phospho-dead TSC1 2A mutant (2A). n = 96 individual cells. (K) Schematic model of the TSC1-mTORC1-TSC1 negative feedback loop. Elevated lysosomal mTORC1 activity drives TSC1 phosphorylation in specific residues that prevents its proteasome-mediated degradation. In turn, phosphorylated TSC1 preferentially downregulates lysosomal mTORC1 signaling, thus activating TFEB-dependent lysosome biogenesis (see also text for details). Data in graphs shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The remaining volume of the supernatants was incubated with 1 μL anti-TSC1 antibody (#6935, Cell Signaling Technology) at 4 °C in an overhead rotator for 3 h, followed by incubation with 30 μL pre-washed Protein A agarose bead slurry (#11134515001, Roche) for an additional hour at 4 °C in an overhead rotator.

Techniques: Western Blot, Expressing, Mutagenesis, Control, Immunoprecipitation, Blocking Assay, Phospho-proteomics, Confocal Microscopy, Activity Assay

Journal: Frontiers in Immunology

Article Title: TSC1 deficiency drives immune evasion in colorectal cancer via mTORC1-mediated dysregulation of PD-L1 sialylation

doi: 10.3389/fimmu.2025.1692210

Figure Lengend Snippet: Immune infiltration analysis of TSC1 in colorectal cancer. (A) Immune Infiltration Landscape. (B) Comparative analysis of immune infiltration across experimental groups. (C) Association of TSC1 expression with distinct immune cell populations.

Article Snippet: Tissue sections were incubated overnight at 4 °C with the following primary antibodies: anti-TSC1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 6935S), anti-PD-L1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 13684S), anti-ST6GALNAC1 goat polyclonal antibody (1:100, Invitrogen, PA5-31200), and biotinylated Sambucus Nigra Lectin (SNA, 1:500, Vector Laboratories, B-1305-2) to detect α-2,6 sialylated glycoconjugates.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: TSC1 deficiency drives immune evasion in colorectal cancer via mTORC1-mediated dysregulation of PD-L1 sialylation

doi: 10.3389/fimmu.2025.1692210

Figure Lengend Snippet: TSC1 deficiency promotes proliferation and migration of colon cancer cells. (A) Immunofluorescence staining of TSC1 in normal and cancerous tissues. (B) Immunofluorescence staining of ST6GALNAC1 and PD-L1 in normal and cancerous tissues. (C) Analysis of sialylation patterns across normal mucosa, adenomatous polyps, and malignant tissues. (D) Wound healing assay to assess cell proliferation after TSC1 knockdown. (E) Representative images of crystal violet-stained migrated cells. Data are presented as mean ± SEM (n=3). *** p < 0.001, ** p < 0.01, * p < 0.05 vs control group.

Article Snippet: Tissue sections were incubated overnight at 4 °C with the following primary antibodies: anti-TSC1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 6935S), anti-PD-L1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 13684S), anti-ST6GALNAC1 goat polyclonal antibody (1:100, Invitrogen, PA5-31200), and biotinylated Sambucus Nigra Lectin (SNA, 1:500, Vector Laboratories, B-1305-2) to detect α-2,6 sialylated glycoconjugates.

Techniques: Migration, Immunofluorescence, Staining, Wound Healing Assay, Knockdown, Control

Journal: Frontiers in Immunology

Article Title: TSC1 deficiency drives immune evasion in colorectal cancer via mTORC1-mediated dysregulation of PD-L1 sialylation

doi: 10.3389/fimmu.2025.1692210

Figure Lengend Snippet: TSC1 deficiency remodels sialic acid metabolic homeostasis via the mTORC1-S6K1 signaling pathway and promotes tumor immune escape. (A) qPCR analysis of ST6GALNAC1, Neu4, and NPL mRNA expression. (B) Representative Western blot images showing the expression levels of ST6GALNAC1, Neu4, and PD-L1 in indicated groups. (C) Analysis of PD-L1 sialylation levels by lectin blot assay. (D) Assessment of CD8+ T cell cytotoxic function. Data are presented as mean ± SEM (n=3). *** p < 0.001, ** p < 0.01, * p < 0.05 vs control group.

Article Snippet: Tissue sections were incubated overnight at 4 °C with the following primary antibodies: anti-TSC1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 6935S), anti-PD-L1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 13684S), anti-ST6GALNAC1 goat polyclonal antibody (1:100, Invitrogen, PA5-31200), and biotinylated Sambucus Nigra Lectin (SNA, 1:500, Vector Laboratories, B-1305-2) to detect α-2,6 sialylated glycoconjugates.

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: TSC1 deficiency drives immune evasion in colorectal cancer via mTORC1-mediated dysregulation of PD-L1 sialylation

doi: 10.3389/fimmu.2025.1692210

Figure Lengend Snippet: Animal experiments validate the immunosuppressive microenvironment after TSC1 knockdown. (A) Tumor volume and weight in the TSC1 knockdown model treated with anti-PD-1 therapy. (B) TSC1 knockdown MC38 cell subcutaneous xenograft model. Data are presented as mean ± SEM (n=3). *** p < 0.001, * p < 0.05 vs control group.

Article Snippet: Tissue sections were incubated overnight at 4 °C with the following primary antibodies: anti-TSC1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 6935S), anti-PD-L1 rabbit monoclonal antibody (1:200, Cell Signaling Technology, 13684S), anti-ST6GALNAC1 goat polyclonal antibody (1:100, Invitrogen, PA5-31200), and biotinylated Sambucus Nigra Lectin (SNA, 1:500, Vector Laboratories, B-1305-2) to detect α-2,6 sialylated glycoconjugates.

Techniques: Knockdown, Control