Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Schematic depicting how Tsc1 WT and KO MEFs were acquired and the OSKM reprogramming procedure. ( B ) Western blot showing TSC1, TFE3 protein levels, and mTORC1 activity on day 5 in Tsc1 WT and KO MEFs transduced with OSKM, and either Ctrl or Tfe3 shRNAs. D day, hereafter in all similar experiments. ( C ) Images and quantification of AP + colonies (left panel) and phase contrast or NANOG-GFP + colonies (right panel) at day 12 in Tsc1 WT and KO MEFs transduced with OSKM ( n = 3 each with biological replicates). Scale bar: 100 µm. ( D ) The number of differentially expressed genes (DEGs) in Tsc1 WT and KO MEFs transduced with OSKM at day 5 (D5) and day 10 (D10). Fold change (FC) >1.5 and P value <0.05. ( E ) Venn diagram showing the overlap of upregulated and downregulated genes from OSKM day 5 and day 10 in Tsc1 KO cells compared with WT cells. ( F ) Gene ontology (GO) analysis of upregulated (UP) and downregulated (DOWN) genes in Tsc1 KO cells compared with WT cells at OSKM day 5 and day 10. ( G ) Discovery of known motifs from differentially expressed genes (DEGs) at day 5 and day 10 in Tsc1 KO compared to WT MEFs transduced with OSKM as in ( D ). ( H ) Fluorescence images showing DAPI and TFE3 localization (left) and quantification of nuclear (N)/cytoplasmic (C) TFE3 ratios (right) at day 5 in Tsc1 WT and KO MEFs transduced with OSKM ( n = 2 each with biological replicates, each replicate of data was from more than 100 cells). Scale bar: 10 µm. ( I ) Western blot showing H3 (nuclear marker), TUBULIN (cytoplasmic marker) and TFE3 protein levels on day 5 in nuclear and cytoplasmic extracts of Tsc1 WT, KO, and KOR (KO + rapamycin) MEFs transduced with OSKM. ( J ) Western blot showing TSC1, TFE3 protein levels, and mTORC1 activity on day 5 in Tsc1 WT and KO MEFs transduced with OSKM, and either Ctrl or Tfe3 shRNAs. ( K ) Phase contrast or NANOG immunofluorescent images and quantification of NANOG-GFP + colonies at day 12 in Tsc1 WT and KO MEFs transduced with OSKM and either Ctrl or Tfe3 shRNAs ( n = 3 each with biological replicates). Scale bar: 100 µm. Data information: In ( C , K ), data are presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t -test). In ( F ), Gene Ontology (GO) analysis for RNA-seq data was performed at the Gene Ontology Resource website. .

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Western Blot, Activity Assay, Transduction, Fluorescence, Marker, RNA Sequencing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) PCA analysis of the indicated samples. RNA-seq data from Florian Villegas et al, . ( B ) Hierarchical clustering of RNA-seq data for DEGs, corresponding GO analysis at the indicated samples as in ( A ). Fold change (FC) >1.5 and P value <0.05. ( C ) TFE3 interactome overlap was observed between proteins upregulated with TFE3 nuclear localization (Ectopic TFE3 binding) and those downregulated upon Tfe3 knockout (endogenous TFE3 binding). Fold change (FC) >2, P value <0.05. ( D ) Co-immunoprecipitation using IgG or TFE3 antibody in extracts from TFE3-ERT (+Tam) 2iL mESCs, followed by Western blotting with the indicated antibodies. ( E ) Schematic depicting how Tsc1 WT and KO OG2 mESCs acquire and pluripotency exit procedure. ( F ) Western blot showing H3, TUBULIN and TFE3 protein levels in nuclear and cytoplasmic extracts of WT, KO, and KOR (KO + rapamycin, hereafter in all similar experiments) mESCs. ( G ) Co-immunoprecipitation using TFE3 antibody in nuclear extracts of WT, KO, and KOR cells after N2B27 40 h exit, followed by Western blotting with the indicated antibodies. ( H ) Knockdown efficiency of Gatad2a and Mbd3 in 2iL mESCs ( n = 3 each with biological replicates). ( I ) Images and quantification of OCT4-GFP + colonies at 72 h in Tsc1 WT and KO cells transduced with Ctrl, Gatad2a or Mbd3 shRNAs in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. ( J ) Images and quantification of OCT4-GFP + colonies at 72 h in mESCs transduced with FLAG or TFE3-ERT in combination with Ctrl, Gatad2a , or Mbd3 shRNAs in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. Data information: In ( H – J ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, and * P < 0.05, n.s. not significant (Student’s t -test). In ( B ), Gene Ontology (GO) analysis for RNA-seq data were performed at the Gene Ontology Resource website. .

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: RNA Sequencing, Binding Assay, Knock-Out, Immunoprecipitation, Western Blot, Knockdown, Transduction

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Western blot showing TSC1 protein level and mTORC1 activity in Tsc1 WT, KO and KOR (KO + rapamycin, also hereafter in all similar experiments) mESCs in 2iL medium. ( B ) Fluorescence images showing DAPI and TFE3 localization (left) and quantification of nuclear (N)/cytoplasmic ( C ) TFE3 ratios (right) in Tsc1 WT, KO, and KOR mESCs in 2iL medium ( n = 2 each with biological replicates, each replicate of data were from more than 100 cells). Scale bar: 10 µm. ( C ) Western blot showing TSC1, TFE3 protein levels, and mTORC1 activity in Tsc1 WT and KO mESCs transduced with either Ctrl or Tfe3 shRNAs. ( D ) Images and quantification of OCT4-GFP + colonies at 72 h in Tsc1 WT and KO mESCs transduced with either Ctrl or Tfe3 shRNAs in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. ( E ) Fluorescence images showing DAPI and TFE3 localization (left) and quantification of nuclear (N)/cytoplasmic (C) TFE3 ratios (right) in mESCs transduced with TFE3-ERT in 2iL medium with or without Tam ( n = 2 each with biological replicates, each replicate of data were from more than 100 cells). Scale bar: 10 µm. ( F ) Images and quantification of OCT4-GFP + colonies at 72 h in mESCs transduced with FLAG or TFE3-ERT in 2iL medium with or without Tam, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. ( G ) Images and quantification of OCT4-GFP + colonies at 72 h in Tsc1 KO and WT mESCs transduced with Ctrl, Gatad2a or Mbd3 shRNAs in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. ( H ) Images and quantification of OCT4-GFP + colonies at 72 h in Tsc1 KO and WT mESCs treated with Ctrl or 0.5 mM NaB in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. ( I ) Images and quantification of OCT4-GFP + colonies at 72 h in mESCs transduced with FLAG or TFE3-ERT (+Tam) treated with Ctrl or 0.5 mM NaB in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. Data information: In ( D , F – I ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t -test).

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Western Blot, Activity Assay, Fluorescence, Transduction

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Phase contrast and OCT4-GFP immunofluorescent images of Tsc1 WT and KO mESCs transduced with either Ctrl or Tfe3 shRNAs in N2B27 medium for 96 h. Scale bar: 100 µm. ( B ) Phase contrast and OCT4-GFP immunofluorescent images of mESCs transduced with FLAG and TFE3-ERT in N2B27 medium for 96 h with or without Tam. Scale bar: 100 µm. ( C ) Phase contrast and OCT4-GFP immunofluorescent images of Tsc1 KO and WT mESCs transduced with Ctrl, Gatad2a or Mbd3 shRNAs in N2B27 medium for 96 h. Scale bar: 100 µm. ( D ) Phase contrast and OCT4-GFP immunofluorescent images of Tsc1 WT and KO mESCs transduced with Ctrl, Gatad2a or Mbd3 shRNAs in N2B27 medium for 96 h. Scale bar: 100 µm. ( E ) Phase contrast and OCT4-GFP immunofluorescent images of mESCs transduced with FLAG or TFE3-ERT in combination with Ctrl, Gatad2a and Mbd3 shRNAs in N2B27 medium for 96 h with or without Tam. Scale bar: 100 µm. ( F ) Phase contrast and OCT4-GFP immunofluorescent images of Tsc1 WT and KO mESCs treated with Ctrl or 0.5 mM NaB in N2B27 medium for 96 h. Scale bar: 100 µm. ( G ) Phase contrast and OCT4-GFP immunofluorescent images of mESCs transduced with FLAG or TFE3-ERT (+Tam) treated with Ctrl or 0.5 mM NaB in N2B27 medium for 96 h. Scale bar: 100 µm. ( H ) Phase contrast and OCT4-GFP immunofluorescent images of mESCs transduced with FLAG or TFE3-ERT in combination with Ctrl or LIN28A overexpression in N2B27 medium for 96 h. Scale bar: 100 µm.

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Transduction, Over Expression

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Heatmaps showing naïve (top) and primed (bottom) pluripotency gene expression during exit (34 h) in mESCs transduced with FLAG or TFE3-ERT (+Tam). RNA-seq data from Florian Villegas et al, . ( B ) RT-qPCR detection of Nanog , Esrrb and Klf4 expression levels in 2iL mESCs and Tsc1 WT, KO mESCs transduced with Ctrl, Gatad2a, or Mbd3 shRNAs in N2B27 medium for 96 h ( n = 3 each with biological replicates). ( C ) RT-qPCR detection of Fgf5 and Zic3 expression levels in 2iL mESCs and Tsc1 WT, KO mESCs transduced with Ctrl, Gatad2a or Mbd3 shRNAs in N2B27 medium for 96 h ( n = 3 each with biological replicates). Data information: In ( B , C ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t- test).

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Gene Expression, Transduction, RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Heatmap of TFE3 (left), RBBP4 (right) and IgG binding in Tsc1 WT and KO mESCs under 2iL conditions. ( B ) Venn diagram showing the overlap of genes with increased binding of TFE3 and RBBP4 in Tsc1 KO 2iL mESCs and downregulated DEGs in TFE3-ERT 34 h exit mESCs (Fig. , clusters 1 and 2) (top) and corresponding GO analysis (bottom). ( C ) Selected genomic views of TFE3, RBBP4 and IgG binding sites in 2iL condition for the indicated genes in Tsc1 WT and KO mESCs. ( D ) Western blot showing TSC1 and LIN28A protein levels in Tsc1 WT and KO mESCs. ( E ) Western blot showing TFE3 and LIN28A protein levels in mESCs transduced with FLAG or TFE3-ERT in 2iL medium with or without Tam. ( F ) RT-qPCR detection of exogenous Lin28a expression levels in 2iL mESCs transduced with FLAG or TFE3-ERT with or without Tam ( n = 3 each with biological replicates). ( G ) Images and quantification of OCT4-GFP + colonies at 72 h in mESCs transduced with FLAG or TFE3-ERT in combination with Ctrl or LIN28A overexpression in 2iL medium, after initial culture in N2B27 medium for 96 h ( n = 3 each with biological replicates). Scale bar: 100 µm. Data information: In ( F , G ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t -test). In ( B ), Gene Ontology (GO) analysis for RNA-seq data was performed at the Gene Ontology Resource website. .

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Binding Assay, Western Blot, Transduction, Quantitative RT-PCR, Expressing, Over Expression, RNA Sequencing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Heatmap of TFE3 (left) and RBBP4 (right) binding in Tsc1 WT and KO mESCs under N2B27 40 h conditions. ( B ) Venn diagram showing the overlap of genes with increased binding of TFE3 and RBBP4 in Tsc1 KO N2B27 40 h exit mESCs and downregulated DEGs in TFE3-ERT 34 h exit mESCs (Fig. , clusters 1 and 2) (top) and corresponding GO analysis (bottom). ( C ) Selected genomic views of TFE3 and RBBP4 binding sites for the indicated genes in Tsc1 WT and KO N2B27 40 h exit mESCs. ( D ) Venn diagram showing the overlap of genes with increased binding of TFE3 and RBBP4 in Tsc1 KO mESCs between 2iL and N2B27 40 h exit conditions. ( E ) Venn diagrams showing the overlap between TFE3 and RBBP4 binding sites in Tsc1 WT and KO (top) and corresponding GO analysis (bottom) under 2iL conditions. ( F ) Venn diagrams showing the overlap between TFE3 and RBBP4 binding sites in Tsc1 WT and KO (top) and corresponding GO analysis (bottom) under N2B27 40 h exit conditions. Data information: In ( B , D – F ), Gene Ontology (GO) analysis for RNA-seq data was performed at the Gene Ontology Resource website.

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Binding Assay, RNA Sequencing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Co-immunoprecipitation using IgG or TFE3 antibody in extracts from OSKM-induced reprogramming TFE3-ERT (+Tam) MEFs at day 5, followed by Western blotting with the indicated antibodies. ( B ) Co-immunoprecipitation using TFE3 antibody in nuclear extracts from WT, KO and KOR OSKM-induced reprogramming cells, followed by Western blotting with the indicated antibodies. ( C ) Knockdown efficiency of Gatad2a and Mbd3 shRNAs at day 5 in MEFs transduced with OSKM and either Ctrl or Tsc2 shRNAs ( n = 3 each with biological replicates). ( D ) Images and quantification of phase contrast or OCT4-GFP immunofluorescent images at day 12 in MEFs transduced with OSKM and Ctrl, or Tsc2 in combination with Ctrl, Gatad2a or Mbd3 shRNAs ( n = 3 each with biological replicates). Scale bar: 100 µm. ( E ) Images and quantification of phase contrast or OCT4-GFP immunofluorescent images at day 12 in MEFs transduced with OSKM and either FLAG, or TFE3-ERT in combination with Ctrl, Gatad2a and Mbd3 shRNAs in medium with Tam ( n = 3 each with biological replicates). Scale bar: 100 µm. ( F ) PCA of the specified samples at day 5/10 in Tsc1 WT and KO MEFs transduced with OSKM and either Ctrl or Tfe3 shRNAs. MEF data from GSE93027 , iPSCs data from GSE93027 , ESCs data from GSE93027 . ( G ) Hierarchical clustering of RNA-seq data for DEGs and corresponding Gene Ontology (GO) analysis at day 5/10 in Tsc1 WT and KO MEFs transduced with OSKM and either Ctrl or Tfe3 shRNAs. Fold change (FC) >2, P value <0.05. ( H ) RT-qPCR detection of Nanog and Esrrb expression levels at day 12 in MEFs transduced with OSKM and either Ctrl, Tsc2 or Tfe3 shRNAs ( n = 3 each with biological replicates). ( I ) RT-qPCR detection of Nanog and Esrrb expression levels at day 12 in MEFs transduced with OSKM and Ctrl, or Tsc2 in combination with Ctrl, Gatad2a or Mbd3 shRNAs ( n = 3 each with biological replicates). Data information: In ( C – E , H , I ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t -test). In ( G ), Gene Ontology (GO) analysis for RNA-seq data were performed at the Gene Ontology Resource website. .

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Immunoprecipitation, Western Blot, Knockdown, Transduction, RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Phase contrast or OCT4-GFP immunofluorescent images and quantification of OCT4-GFP+ colonies at day 12 in MEFs transduced with OSKM and either Ctrl or Tsc2 shRNAs in medium supplemented with Ctrl, 0.3 mM NaB or 1 mM NaB ( n = 3 each with biological replicates). Scale bar: 100 µm. ( B ) Phase contrast or OCT4-GFP immunofluorescent images and quantification of OCT4-GFP+ colonies at day 12 in MEFs transduced with OSKM and either FLAG or TFE3-ERT in medium with Tam, and either Ctrl or 0.3 mM NaB ( n = 3 each with biological replicates). Scale bar: 100 µm. ( C ) Heatmaps showing naïve (left) and primed (right) pluripotency gene expression at day 5 and day 10 in Tsc1 WT and KO MEFs transduced with OSKM and either Ctrl or Tfe3 shRNAs. ( D ) Venn diagrams showing the overlap between TFE3 and RBBP4 binding sites in Tsc1 WT, KO and KOR (top) and corresponding GO analysis (bottom). Data information: In ( A , B ), data were presented as mean values ± SD. *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant (Student’s t -test). In ( D ), Gene Ontology (GO) analysis for RNA-seq data was performed at the Gene Ontology Resource website.

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Transduction, Gene Expression, Binding Assay, RNA Sequencing

Journal: EMBO Reports

Article Title: Hyperactivation of mTORC1 blocks stem cell fate transitions through TFE3-NuRD association

doi: 10.1038/s44319-025-00544-z

Figure Lengend Snippet: ( A ) Heatmap illustrating the density of TFE3(left), RBBP4(right) and IgG CUT&Tag reads on day 10 in Tsc1 WT, KO, and KOR MEFs transduced with OSKM. ( B ) Venn diagram showing the overlap of genes with increased binding of TFE3 and RBBP4 and downregulated DEGs in Tsc1 KO reprogrammed cells (Fig. , cluster 1). ( C ) GO analysis of 386 genes from ( B ). ( D ) Selected genomic views of TFE3, RBBP4 and IgG binding sites for the indicated genes at day 10 in Tsc1 WT, KO and KOR MEFs transduced with OSKM. ( E ) Schematic diagram illustrating how the mTORC1-TFE3-NuRD axis controls pluripotent stem cells homeostasis maintenance cell fate transitions. Data information: In ( G ), Gene Ontology (GO) analysis for RNA-seq data was performed at the Gene Ontology Resource website. .

Article Snippet: Tsc1 Loxp/Loxp mice , The Jackson Laboratory , 005680.

Techniques: Transduction, Binding Assay, RNA Sequencing

Journal: iScience

Article Title: Regulatory role of mTORC1 signaling in osteoblasts in acute myeloid leukemia progression and steady-state hematopoiesis

doi: 10.1016/j.isci.2025.114533

Figure Lengend Snippet: mTORC1 activation in osteoblasts accelerates AML progression (A) Schematic diagram of generation of tissue-specific Tsc1 knockout mice. (B) Schematic diagram of generation of a MLL-AF9 murine AML model. (C and D) Representative flow cytometry plots and percentages of GFP + AML cells in the (C) peripheral blood ( n = 7–9) and (D) bone marrow ( n = 11) of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice. (E–G) Representative flow cytometry plots and percentages of (E) GFP + c-Kit – AML cells and GFP + c-Kit + AML cells ( n = 6–7), (F) BrdU + GFP + c-Kit + AML cells ( n = 4–5), and (G) Annexin V + GFP + c-Kit + AML cells ( n = 4–5) in the bone marrow of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice. (H) Percentages of GFP + AML cells in the peripheral blood and bone marrow of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice 8 days after the second transplantation ( n = 3). (I and J) Survival probabilities of (I) Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl AML mice ( n = 6–7) and (J) WT AML mice transplanted with c-Kit + AML cells from Raptor fl/fl and Col1a1-Cre;Raptor fl/fl mice ( n = 5–7). All mice used in this study were male. n.s., not significant. ∗ p < 0.05. Error bars show the standard deviation.

Article Snippet: Tsc1 fl/fl mice , Jackson Laboratory , #005680.

Techniques: Activation Assay, Knock-Out, Flow Cytometry, Transplantation Assay, Standard Deviation

Journal: iScience

Article Title: Regulatory role of mTORC1 signaling in osteoblasts in acute myeloid leukemia progression and steady-state hematopoiesis

doi: 10.1016/j.isci.2025.114533

Figure Lengend Snippet: Activation of mTORC1 in osteoblasts impairs normal hematopoiesis (A) Schematic diagram of flow cytometric analysis of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice. (B) The number of BM-MNCs in hind legs of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice ( n = 9–10). (C–F) Representative flow cytometry plots and percentages of (C) LSK cells and LSK subpopulations ( n = 17–18), (D) LK cells and LK subpopulations ( n = 14–15), (E) CD11b + Gr-1 + myeloid cells ( n = 15–16), and (F) B220 + IgM – immature and B220 + IgM + mature B cells ( n = 16–18) in the bone marrow of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice. (G) Representative flow cytometry plots and fold changes in the percentage of LSK cells ( n = 7–18), CD11b + Gr-1 + myeloid cells ( n = 5–18), and B220 + IgM – immature and B220 + IgM + mature B cells ( n = 5–16) in the bone marrow of Tsc1 fl/fl , Col1a1-Cre;Tsc1 fl/fl , and Col1a1-Cre;Tsc1 fl/fl ;Raptor fl/+ mice. All mice used in this study were male. n.s., not significant. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, # p < 0.05, ## p < 0.01, and ### p < 0.001. Error bars show the standard deviation.

Article Snippet: Tsc1 fl/fl mice , Jackson Laboratory , #005680.

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Journal: iScience

Article Title: Regulatory role of mTORC1 signaling in osteoblasts in acute myeloid leukemia progression and steady-state hematopoiesis

doi: 10.1016/j.isci.2025.114533

Figure Lengend Snippet: IL-6 in osteoblasts plays a crucial role for AML progression (A) Schematic diagram of three datasets used to screen for factors involved in the cell-cell interaction between mTORC1-activated osteoblasts and AML cells. (B and C) Volcano plots showing DEGs (B) in osteoblasts co-cultured with AML cells compared to monocultured osteoblasts and (C) in mTORC1 high osteoblasts compared to mTORC1 low osteoblasts. (D) Venn diagram highlighting overlapping genes identified across the three datasets. (E) Il6 mRNA expression in calvarial osteoblasts from Col1a1-Cre;Tsc1 fl/fl mice ( n = 4–5). (F) GSEA results for the HALLMARK_IL6_JAK_STAT3_SIGNALING gene set in AML cells co-cultured with osteoblasts. (G) MFI of phosphorylated STAT3 in AML cells in the murine AML model and in lineage – cells from WT mice ( n = 4–5). (H) Schematic diagram of generation of the IL-6R-knockdown MLL-AF9 murine AML model. (I) Survival probabilities of Tsc1 fl/fl and Col1a1-Cre;Tsc1 fl/fl mice transplanted with shCtrl- or sh Il6r -transduced AML cells ( n = 10–20). (J) Schematic model of the findings of this study. Osteoblastic mTORC1 signaling enhances IL-6 production, which activates JAK/STAT3 signaling to promote cell proliferation and inhibit apoptosis in undifferentiated AML cells, driving AML progression. All mice used in this study were male. n.s., not significant. ∗ p < 0.05, ∗∗∗ p < 0.001, and # p < 0.05. Error bars show the standard deviation.

Article Snippet: Tsc1 fl/fl mice , Jackson Laboratory , #005680.

Techniques: Cell Culture, Expressing, Knockdown, Standard Deviation

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