tsc1  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: Cerebellar acceleration of learning in an evidence-accumulation task

doi: 10.1016/j.celrep.2026.117262

Figure Lengend Snippet: (A) L7-Tsc1 mutant mice have reduced numbers of Purkinje cells in the cerebellar cortex. Scale bars, 100 μm. (B) Schematic of sagittal and coronal views of the cerebellum with quantification of Purkinje cell loss averaged over four L7-Tsc1 mutant mice at 5–6 months old for each cerebellar lobule, normalized to three wild-type littermates. (C) Kaplan-Meier estimator of probability of reaching the final level of task training for L7-Tsc1 mutant mice (log-rank test, ** p ≤ 0.01). Shaded areas in Kaplan-Meier curves represent 95% CIs. (D) Ternary plots for the composition of latent states with 95% confidence bands (shaded regions) around the compositional mean (open symbols) for L7-Tsc1 mutant mice and control mice (wild-type littermates). Each data point represents one mouse. The 95% confidence regions are based on an assumption of normality for compositions. Arrows indicate the shift from the average of the control group to the average of the L7-Tsc1 mutant mice. (E) Psychometric performance curves in mice who recently reached the final level show no detectable change in bias, slope, or lapse rate. Thick lines indicate averages; thin lines are data from individual animals. Data from all eight L7-Tsc1 mutant mice are included as well as 17 out of 23 control mice (six control animals did not reach the final version of the task). (F) Left: sensory sensitivity test with bilateral and unilateral whisker puffs in wild-type C57BL/6J mice. Right: median number of blinks in response to puffs of different durations for L7-Tsc1 mutant mice ( n = 16) and wild-type littermates ( n = 7; two-way ANOVA, ** p ≤ 0.01). Shaded areas indicate the estimated SEM. (G) Increased salience through stronger puffs leads to enhanced learning (log-rank test, *** p ≤ 0.001). Kaplan-Meier estimator of task completion for animals receiving standard (10 psi, nine mice) or stronger (20 psi, nine mice) puffs. Shaded areas represent 95% CIs. (H) Ternary compositional plot of latent states with 95% confidence bands (shaded regions) around the compositional mean (open symbols) at middle levels of training. The arrow indicates the shift from the 10-psi average to the group receiving stronger airpuffs. (I) L7-Tsc1 mutant mice express an island of enhanced sensory reactivity and task focus amid a variety of impairments. Each dot represents an estimated effect size (Cohen’s d ) for the behavior of L7-Tsc1 mutant mice compared to their wild-type littermates. The thick circle indicates typical behavior (effect size 0). Results are based on data presented in this paper and from Tsai et al., Kloth et al., and Klibaite et al.

Article Snippet: To create these Purkinje cell specific L7 Cre ; Tsc1 flox/flox mice, Tsc1 flox/flox ( Tsc1 tm1Djk /J, The Jackson Laboratory stock #005680) mutant mice were crossed into L7-Cre mice (B6.129-Tg(Pcp2-cre)2Mpin/J, The Jackson Laboratory, stock #004146).

Techniques: Mutagenesis, Control, Whisker Assay

Journal: Cell reports

Article Title: Cerebellar acceleration of learning in an evidence-accumulation task

doi: 10.1016/j.celrep.2026.117262

Figure Lengend Snippet: (A) Schematic of silicon probe recording site in forebrain from mice receiving cue-locked optogenetic stimulation of crus I. (B) Average firing rates in anterior cingulate cortex. The addition of optogenetic stimulation resulted in time-shifted responses to airpuffs. (C) Cerebellar recording site in the whisker puffs + cue-locked optogenetic experiment. (D) Example raster plots of Purkinje cell complex spikes during 20 trials with only a whisker puff (top) or with a whisker puff paired with cue-locked optogenetic stimulation (middle). Bottom: Purkinje cell complex-spike recordings (average data from four mice) show that pairing airpuffs with optogenetic stimuli led to a shift in the time course of activity toward later times after airpuff onset. (E) Schematic of silicon probe recording site in forebrain from L7-Tsc1 mutant mice. (F) Left: increased spontaneous in vivo firing rates in the anterior cingulate cortex in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Right: average firing rates in anterior cingulate cortex in response to an airpuff to the whiskers (four mutant and five wild-type mice). Responses varied somewhat by genetic background (compare with B and J). (G) Cerebellar recording site from L7-Tsc1 mutant mice. (H) Left: reduced spontaneous in vivo firing rates of complex spikes in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Above the plot is an example waveform of a complex spike; total duration of the example trace is 15 ms. Right: example raster plots of Purkinje cell complex spikes during 20 trials from one wild-type animal (top) and one L7-Tsc1 mutant animal (bottom). Bottom right: Delayed time course of the airpuff evoked response of complex spikes compared with wild-type littermates. (I) Schematic of silicon probe recording site in forebrain from mice receiving continuous bilateral optogenetic stimulation of crus I. (J) Average firing rates in anterior cingulate cortex. The addition of optogenetic stimulation reduced responses to airpuffs. (K) Cerebellar recording site in the whisker puffs + continuous optogenetic experiment. (L) Example raster plots of Purkinje cell complex spikes during 20 trials with only a whisker puff (top) or with a whisker puff paired with continuous optogenetic stimulation (middle). Bottom: Purkinje cell complex-spike recordings show that pairing airpuffs with optogenetic stimuli led to a shift in the time course of activity toward later times after airpuff onset. (M) Simple spikes in the Purkinje cells stimulated with cue-locked optogenetic activation led to an increased firing rate without time shift. (N) Left: reduced spontaneous in vivo firing rates of simple spikes in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Above the plot is an example waveform of a simple spike; total duration of the example trace is 15 ms. Right: delayed and smaller simple-spike response in mutant mice compared with wild-type littermates. (O) Simple spikes in the Purkinje cells stimulated with continuous bilateral optogenetic activation led to an increased overall firing rate without a specific response to the airpuff. In (B), (D), (F), (H), (J), (L), (M), (N), and (O) shaded areas represent 95% confidence intervals.

Article Snippet: To create these Purkinje cell specific L7 Cre ; Tsc1 flox/flox mice, Tsc1 flox/flox ( Tsc1 tm1Djk /J, The Jackson Laboratory stock #005680) mutant mice were crossed into L7-Cre mice (B6.129-Tg(Pcp2-cre)2Mpin/J, The Jackson Laboratory, stock #004146).

Techniques: Whisker Assay, Activity Assay, Mutagenesis, In Vivo, Two Tailed Test, Activation Assay

Journal: Cell reports

Article Title: Cerebellar acceleration of learning in an evidence-accumulation task

doi: 10.1016/j.celrep.2026.117262

Figure Lengend Snippet: (A) L7-Tsc1 mutant mice have reduced numbers of Purkinje cells in the cerebellar cortex. Scale bars, 100 μm. (B) Schematic of sagittal and coronal views of the cerebellum with quantification of Purkinje cell loss averaged over four L7-Tsc1 mutant mice at 5–6 months old for each cerebellar lobule, normalized to three wild-type littermates. (C) Kaplan-Meier estimator of probability of reaching the final level of task training for L7-Tsc1 mutant mice (log-rank test, ** p ≤ 0.01). Shaded areas in Kaplan-Meier curves represent 95% CIs. (D) Ternary plots for the composition of latent states with 95% confidence bands (shaded regions) around the compositional mean (open symbols) for L7-Tsc1 mutant mice and control mice (wild-type littermates). Each data point represents one mouse. The 95% confidence regions are based on an assumption of normality for compositions. Arrows indicate the shift from the average of the control group to the average of the L7-Tsc1 mutant mice. (E) Psychometric performance curves in mice who recently reached the final level show no detectable change in bias, slope, or lapse rate. Thick lines indicate averages; thin lines are data from individual animals. Data from all eight L7-Tsc1 mutant mice are included as well as 17 out of 23 control mice (six control animals did not reach the final version of the task). (F) Left: sensory sensitivity test with bilateral and unilateral whisker puffs in wild-type C57BL/6J mice. Right: median number of blinks in response to puffs of different durations for L7-Tsc1 mutant mice ( n = 16) and wild-type littermates ( n = 7; two-way ANOVA, ** p ≤ 0.01). Shaded areas indicate the estimated SEM. (G) Increased salience through stronger puffs leads to enhanced learning (log-rank test, *** p ≤ 0.001). Kaplan-Meier estimator of task completion for animals receiving standard (10 psi, nine mice) or stronger (20 psi, nine mice) puffs. Shaded areas represent 95% CIs. (H) Ternary compositional plot of latent states with 95% confidence bands (shaded regions) around the compositional mean (open symbols) at middle levels of training. The arrow indicates the shift from the 10-psi average to the group receiving stronger airpuffs. (I) L7-Tsc1 mutant mice express an island of enhanced sensory reactivity and task focus amid a variety of impairments. Each dot represents an estimated effect size (Cohen’s d ) for the behavior of L7-Tsc1 mutant mice compared to their wild-type littermates. The thick circle indicates typical behavior (effect size 0). Results are based on data presented in this paper and from Tsai et al., Kloth et al., and Klibaite et al.

Article Snippet: Mouse: Tsc1 flox/flox : Tsc1 tm1Djk /J , The Jackson Laboratory , JAX #005680; RRID:IMSR_JAX:005680.

Techniques: Mutagenesis, Control, Whisker Assay

Journal: Cell reports

Article Title: Cerebellar acceleration of learning in an evidence-accumulation task

doi: 10.1016/j.celrep.2026.117262

Figure Lengend Snippet: (A) Schematic of silicon probe recording site in forebrain from mice receiving cue-locked optogenetic stimulation of crus I. (B) Average firing rates in anterior cingulate cortex. The addition of optogenetic stimulation resulted in time-shifted responses to airpuffs. (C) Cerebellar recording site in the whisker puffs + cue-locked optogenetic experiment. (D) Example raster plots of Purkinje cell complex spikes during 20 trials with only a whisker puff (top) or with a whisker puff paired with cue-locked optogenetic stimulation (middle). Bottom: Purkinje cell complex-spike recordings (average data from four mice) show that pairing airpuffs with optogenetic stimuli led to a shift in the time course of activity toward later times after airpuff onset. (E) Schematic of silicon probe recording site in forebrain from L7-Tsc1 mutant mice. (F) Left: increased spontaneous in vivo firing rates in the anterior cingulate cortex in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Right: average firing rates in anterior cingulate cortex in response to an airpuff to the whiskers (four mutant and five wild-type mice). Responses varied somewhat by genetic background (compare with B and J). (G) Cerebellar recording site from L7-Tsc1 mutant mice. (H) Left: reduced spontaneous in vivo firing rates of complex spikes in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Above the plot is an example waveform of a complex spike; total duration of the example trace is 15 ms. Right: example raster plots of Purkinje cell complex spikes during 20 trials from one wild-type animal (top) and one L7-Tsc1 mutant animal (bottom). Bottom right: Delayed time course of the airpuff evoked response of complex spikes compared with wild-type littermates. (I) Schematic of silicon probe recording site in forebrain from mice receiving continuous bilateral optogenetic stimulation of crus I. (J) Average firing rates in anterior cingulate cortex. The addition of optogenetic stimulation reduced responses to airpuffs. (K) Cerebellar recording site in the whisker puffs + continuous optogenetic experiment. (L) Example raster plots of Purkinje cell complex spikes during 20 trials with only a whisker puff (top) or with a whisker puff paired with continuous optogenetic stimulation (middle). Bottom: Purkinje cell complex-spike recordings show that pairing airpuffs with optogenetic stimuli led to a shift in the time course of activity toward later times after airpuff onset. (M) Simple spikes in the Purkinje cells stimulated with cue-locked optogenetic activation led to an increased firing rate without time shift. (N) Left: reduced spontaneous in vivo firing rates of simple spikes in L7-Tsc1 mutant mice (two-tailed Student’s t test, *** p ≤ 0.001). Above the plot is an example waveform of a simple spike; total duration of the example trace is 15 ms. Right: delayed and smaller simple-spike response in mutant mice compared with wild-type littermates. (O) Simple spikes in the Purkinje cells stimulated with continuous bilateral optogenetic activation led to an increased overall firing rate without a specific response to the airpuff. In (B), (D), (F), (H), (J), (L), (M), (N), and (O) shaded areas represent 95% confidence intervals.

Article Snippet: Mouse: Tsc1 flox/flox : Tsc1 tm1Djk /J , The Jackson Laboratory , JAX #005680; RRID:IMSR_JAX:005680.

Techniques: Whisker Assay, Activity Assay, Mutagenesis, In Vivo, Two Tailed Test, Activation Assay

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