Journal: Molecular neurodegeneration

Article Title: Upregulated astrocyte HDAC7 induces Alzheimer-like tau pathologies via deacetylating transcription factor-EB and inhibiting lysosome biogenesis.

doi: 10.1186/s13024-025-00796-2

Figure Lengend Snippet: Fig. 7 HDAC7 regulates TFEB acetylation and lysosomal biogenesis. A Representative blots showing HDAC7-TFEB interaction and TFEB acetylation level by immunoprecipitation conducted in primary astrocytes overexpressing HDAC7 or vector. B Quantification of TFEB acetylation in vector and HDAC7-overexpressing astrocytes. n = 5 per group. C Representative immunostaining images of TFEB in vector and HDAC7-overexpressing astrocytes treated with Torin1 (100 nM) or vehicle for 4 h. Scale bar:10 μm. D Quantification of nuclear/cytoplasmic TFEB ratio in C. n = 31 (Vec), 32 (HDAC7), 38 (Vec+Torin1), 38 (HDAC7+Torin1) cell per group. E Analysis of TFEB transcriptional activity in HEK293T cells transfected with HDAC7 or vector and then treated with Torin1 (100 nM) or vehicle for 4 h. n = 4 per group. F mRNA levels of Lamp1, Ctsb, Ctsd, Ctsf and Map1lc3b measured by RT-qPCR in cultured astrocytes treated as described in C. n = 4 per group. G, H Immunoprecipitation assay showing the acetylation level of TFEB in WT and HDAC7 knockout astrocytes. n = 3 per group. I Representative immunostaining images of TFEB in control and HDAC7 knockout astrocytes. Scale bar: 10 μm. J Quantification of nuclear/cytoplasmic TFEB ratio in I. n = 31 (HDAC7+/+), 37 (HDAC7-/-). K Analysis of TFEB transcriptional activity in HEK293T cells transfected with shRNA targeting HDAC7 (shHD7) or control scramble. n = 4 per group. L mRNA levels of Lamp1, Ctsb, Ctsd, Ctsf, Map1lc3b and Atg9b quantified by RT-qPCR in control and HDAC7 knockout astrocytes. n = 4 per group. M, N Western blotting analysis and quantification of LAMP1, CTSB and CTSD in control and HDAC7 knockout astrocytes. n = 6 per group. Statistical significance was determined by unpaired Student’s t test (B, H, J-L and N) or one-way ANOVA with Tukey’s post hoc analysis (D-F). Data are shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: TMP195 (a selective class IIa HDAC inhibitor), Bafilomycin A1 (a H+-ATPase inhibitor) and Torin1 (a mTORC1 inhibitor) were bought from Selleck (Houston, TX, USA).

Techniques: Immunoprecipitation, Plasmid Preparation, Immunostaining, Activity Assay, Transfection, Quantitative RT-PCR, Cell Culture, Knock-Out, Control, shRNA, Western Blot

Journal: Science immunology

Article Title: Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

doi: 10.1126/sciimmunol.abg0791

Figure Lengend Snippet: Fig. 2. SLC7A5-dependent late endosomal mTORC1 activation in naïve CD4+ T cell responses. (A and B) Naïve CD4+ T cells from young and older healthy individuals were activated with anti-CD3/anti-CD28 beads for 3 days in the presence of vehicle or SLC7A5 inhibitor JPH203 (top). Alternatively, cells were activated with anti-CD3/ anti-CD28 beads for 5 days with the last 2 days in the presence of vehicle or JPH203 (bottom). mTORC1 activities were determined either by Western blotting of p-S6K1 (A) or by flow cytometry of intracellular p-S6RP (S235/S236) (B). Data are shown as one representative experiment (left) and cumulative data of three or four experiments (right). (C) Naïve CD4+ T cells were activated for 3 days followed by treatment or not with an AKT inhibitor for 2 hours before harvesting. Endosomes were isolated and analyzed for in vitro mTORC1 kinase activity toward S6K1. Total cell lysates (T) and endosome isolates (E) were analyzed by immunoblotting for indicated proteins. Data are shown as one representative of three experiments. (D) In vitro mTORC1 kinase activity of endosome isolates in day 5–stimulated naïve CD4+ T cells from one young and one older individual. Data are shown as one representative of three experiments. (E) Cells were stained with anti-EEA1, anti-LAMP1, and anti-mTOR. Confocal images representa- tive of two independent experiments are shown. Scale bars, 5 m. (F and G) mTORC1 activities in day 3– and day 5–stimulated naïve CD4+ T cells from young and older in- dividuals after control or VPS39 silencing. The gray histogram represents isotype control. Comparison by two-tailed paired t test (A, B, F, and G). *P < 0.05 and **P < 0.01.

Article Snippet: The mTORC1 inhibitor Torin 1 (4247, Tocris) was used at a dose of 100 nM.

Techniques: Activation Assay, Western Blot, Flow Cytometry, Isolation, In Vitro, Activity Assay, Staining, Control, Comparison, Two Tailed Test

Journal: Science immunology

Article Title: Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

doi: 10.1126/sciimmunol.abg0791

Figure Lengend Snippet: Fig. 4. Sustained activation of late endosomal mTORC1 prevents PD-1 from lysosomal degradation. (A to D) Naïve CD4+ T cells from young and older individuals were activated with anti-CD3/anti-CD28 beads for 5 days with the last 2 days in the presence of vehicle or indicated inhibitor (B and C). Alternatively, cells were transfected with control or silencing RNA and acti- vated for 5 days (A and D). Representative histograms showing cell surface protein expression of PD-1 (left) and cumulative data of cell surface protein expression (middle) and gene expression (right) of PD-1. (E to G) Cell surface expression (E), intracellular expression (F), and cell surface/intracellular PD-1 expression ratio (G) after control or VPS39 silencing in day 3–stimulated naïve CD4+ T cells from older individuals. (H) Control or VPS39-silenced, day 5–stimulated naïve CD4+ T cells from older individuals were treated with CHX (5 g/ml) to inhibit de novo PD-1 synthesis. Total PD-1 protein normalized to -actin expression is shown as relative to nontreatment. Means ± SEM of three experiments. (I) Longitudinal analysis of cell surface protein expression of PD-1 in naïve CD4+ T cells from 10 young and 10 older individuals. Means ± SEM. (J) PD-1 gene expression comparison between day 5–stimulated young and older naïve CD4+ T cells. The gray histogram represents isotype control. Comparison by two-tailed paired (A to G) or two-tailed unpaired t test (I and J). *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The mTORC1 inhibitor Torin 1 (4247, Tocris) was used at a dose of 100 nM.

Techniques: Activation Assay, Transfection, Control, Expressing, Gene Expression, Comparison, Two Tailed Test

Journal: Science immunology

Article Title: Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

doi: 10.1126/sciimmunol.abg0791

Figure Lengend Snippet: Fig. 6. Inhibition of late endosomal mTORC1 promotes primary CD4+ T cell responses after LCMV infection in vivo. (A to C) A 1 × 104 Tfeb shRNA or control shRNA retrovirally transduced Amcyan+ LCMV–specific naïve SMARTA TCR transgenic CD4+ T cells was adoptively transferred into CD45.2+ naïve recipients followed by infection with LCMV Armstrong. On day 8 after infection, spleens were harvested and analyzed. FACS plots are gated on CD4+ Amcyan+ SMARTA cells. (A) Tfeb protein expression in transduced cells before adoptive transfer. (B) PD-1 expression on day 8 after infection; (C) SMARTA CD4+ T cell numbers per spleen on day 8 after infection. (D to K) Analysis of T cells responses as described in (A) to (C) after adoptive transfer of Vps39 shRNA transduced SMARTA CD4+ T cells. (D) Vps39 protein expression in trans- duced cells before adoptive transfer. (E) Phosphorylation of S6RP on day 8 after infection. (F) PD-1 expression on day 8 after infection. (G) SMARTA CD4+ T cell numbers per spleen on day 8 after infection. (H) Cell apoptosis and proliferation of transferred SMARTA CD4+ T cells on day 8 after infection. (I) Mice infected with LCMV after adoptive transfer of transduced cells were additionally treated with anti–PD-1 Ab (29F.1A12) or control IgG on days 0, 3, and 6 after infection. SMARTA CD4+ T cell num- bers were determined on day 8 after LCMV infection. (J) SMARTA CD4+ T cell numbers in the spleen at day 30 after infection. (K) Longitudinal analysis of SMARTA cells in the spleen of LCMV-infected B6 mice. (L to O) A 1 × 104 Slc7a5 shRNA or control shRNA retrovirally transduced Amcyan+ LCMV–specific naïve SMARTA CD4+ T cells was adoptively transferred into CD45.2+ naïve recipients as in (A) to (C). Phosphorylation of S6RP (L), PD-1 expression (M), SMARTA cell numbers per spleen (N), and cell apoptosis and proliferation (O) of transduced SMARTA CD4+ T cells on day 8 after LCMV infection. Data are pooled from two independent experiments with three to four mice per group (B and C and E to G), representative of two independent experiments with three to five mice per group (A, D, and H to K) or one experiment with five mice per group (L to O). Statistical significance by two-tailed unpaired t test (B and C, E to H, and J to O) or one-way ANOVA followed by Tukey’s multiple comparisons test (I). The gray histograms represent naïve cells. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The mTORC1 inhibitor Torin 1 (4247, Tocris) was used at a dose of 100 nM.

Techniques: Inhibition, Infection, In Vivo, shRNA, Control, Transgenic Assay, Expressing, Adoptive Transfer Assay, Phospho-proteomics, Two Tailed Test

Journal: Science immunology

Article Title: Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals.

doi: 10.1126/sciimmunol.abg0791

Figure Lengend Snippet: Fig. 7. Inhibition of late endosomal mTORC1 augments CD4+ T cell helper responses in vivo. A 1 × 104 Vps39 shRNA or control shRNA retrovirally transduced Amcyan+ naïve SMARTA CD4+ T cells was adoptively transferred into CD45.2+ naïve recipient followed by LCMV infection. On days 8 (A to C) and 30 (E) after infection, spleens were harvested and analyzed. Alternatively, on day 30, immune mice were rechallenged with Lm-gp33 for 6 days (F to H). (A) Number of GC SMARTA TFH cells on day 8 after infection. (B and C) Number of endogenous Fas+ GL-7+ GC B cells (B) and CD138+ IgD− plasma cells (C) in host mice on day 8 after infection. Representative contour plots gated on B220+ CD4− B cells (left) and summary data (right). (D) Anti-LCMV nucleoprotein IgG titers in serum on day 14 after infection. n = 5 mice per group. (E) Numbers of Db LCMV GP33-41 (GP33) tetramer+ cells gated on endogenous CD8+ T cells in the spleen of host mice on day 30 after infection. (F) Numbers of endogenous Db LCMV GP33 tetramer+ CD8+ T cells in the spleen on day 6 after Lm-GP33 challenge. (G) Fold expansion of Db GP33 tetramer+ memory CD8+ T cells upon Lm-GP33 on day 6. (H) Cytokine production by CD8+ T cells harvested on day 6 after infection and restimulated with GP33 peptide in vitro. Data are representative of two independent ex- periments with four to five mice per group (A to E) or one experiment with five mice per group (F to H). Statistical significance by two-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. TNF, tumor necrosis factor–; gMFI, geometric MFI.

Article Snippet: The mTORC1 inhibitor Torin 1 (4247, Tocris) was used at a dose of 100 nM.

Techniques: Inhibition, In Vivo, shRNA, Control, Infection, Clinical Proteomics, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Sensitivity to Vγ9Vδ2TCR T cells is imprinted after single mutations during early oncogenesis

doi: 10.1101/2024.11.19.624272

Figure Lengend Snippet: (A) MCF10a cells were transduced with different ErbB2 variants and co-cultured with either Vγ9Vδ2TCR or HER2-CAR transduced T cells. Tumor cells were pre-treated with the PI3K kinase inhibitor Pictilisib at 2uM overnight. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (B) Furthermore, tumor cells were isolated and stained for BTN2A1 via TCR tetramer staining and (C) BTN3A cell surface expression. (D) Protein expression of HER2, phosphorylated AKT (pAKT) and total AKT in MCF10a mutant lines cultured for 24h in full culture medium (F) or medium without additional growth factors (S) . (E) Multiple tumor cell lines were co-cultured with Vγ9Vδ2TCR T-cells after pre-treatment with either the PI3K kinase inhibitor, AKT inhibitor or MEK inhibitor. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (F) Multiple tumor cell lines were pre-treated with either PI3K kinase inhibitor, AKT inhibitor, mTOR inhibitor Rapamycin or mTOR inhibitor Torin1 and subsequently co-cultured with Vγ9Vδ2TCR T cells. After an overnight co-culture, supernatant was used to determine IFNγ production by the Vγ9Vδ2TCR T cells. (G) Heatmap illustrating the expression of genes from the ‘PI3K_and_AKT_family’ gene list, in the WT-AKPS model, with or without PAM. (H) Heatmap of Pearson’s correlation of the expression of genes from the ‘PI3K_and_AKT_family’ and BTNx genes in the WT-AKPS model, with or without PAM.

Article Snippet: The following inhibitors were used in this study; PI3K inhibitor GDC-0941 (Pictilisib) (SelleckChem, #S1065), pan-AKT inhibitor MK2206 (SelleckChem, #S1078), MEK inhibitor AZD6244 (Selumetinib) (SelleckChem, #S1008), mTORC 1 inhibitor Rapamycin (Sirolimus) (SelleckChem, #S1039), mTORC 1 / 2 inhibitor Torin 1 (SelleckChem, #S2827).

Techniques: Transduction, Cell Culture, Co-Culture Assay, Isolation, Staining, Expressing, Mutagenesis

Journal: Life sciences

Article Title: The effects of Tel2 on cardiomyocyte survival

doi: 10.1016/j.lfs.2019.116665

Figure Lengend Snippet: A: Representative immunoblots of Tel2 in adult mouse cardiomyocytes infected with Ad.Tel2. Overexpression of Tel2 was detected by western blotting with anti-human and anti-mouse Tel2 antibodies. Figure shows representative data among 3 independent experiments. B: Effects of Tel2 overexpression on H2O2-induced cell death in isolated cardiomyocytes. One day after infection with Ad.Tel2 or Ad.GFP, cardiomyocytes were exposed to 20 μM H2O2 for 3 hours. To assess the mTOR signaling pathway, cardiomyocytes were pretreated with either 100 nM of rapamycin (Rap), an mTORC1 inhibitor, or 100 nM of Torin-1, an mTORC1 and mTORC2 inhibitor, 30 min prior to H2O2 treatment. DMSO was used as a vehicle control. Morphological changes in cultured cardiomyocytes were assessed before applying the reagents (pre) and at the end of experiments (post). Live/Dead Assays (Live/Dead), in which live cells stain with calcein AM (green) and nuclei of dead cells stain with ethidium homodimer-1 (red), were used to assess cell viability. This is a set of representative images among four independent experiments. Scale bars, 200 μm. C: Quantitative data of cardiomyocyte cell death. The total numbers of live cytosol (green) and dead nuclei (red) cells were counted in 3 low-power fields. More than 500 cells were counted in each condition. Cell Death % was calculated as dead cells/total cells. n = 6. *; p < 0.05, Ad.GFP + DMSO vs. Ad.Tel2 + DMSO, **; p < 0.01, Ad.Tel2 + DMSO vs. Ad.Tel2 + Torin-1, n.s.; no significance, Ad.Tel2 + DMSO vs. Ad.Tel2 + rapamycin (Rap).

Article Snippet: To assess the mTOR signaling pathway in Tel2-mediated cardiomyocyte cell survival, thirty minutes prior to H2O2 treatment, some cells were given an additional pretreatment, either with 100 nM of rapamycin (Cayman Chemical, Ann Arbor, MI), an mTORC1 inhibitor, or 100 nM of Torin-1 (Selleck Chemicals, Houston, TX), an mTORC1 & mTORC2 inhibitor.

Techniques: Western Blot, Infection, Over Expression, Isolation, Control, Cell Culture, Staining