Journal: Molecular Therapy. Nucleic Acids

Article Title: Altered CELF4 splicing factor enhances pancreatic neuroendocrine tumors aggressiveness influencing mTOR and everolimus response

doi: 10.1016/j.omtn.2023.102090

Figure Lengend Snippet: Influence of CELF4 expression on the functional profile of phosphoprotein of the mTOR pathway (A and B) Unsupervised clustering analysis of phosphorylated protein levels of mTOR pathway components in CELF4 -silenced QGP-1 (A) and BON-1 (B) cells (1; green) compared to scramble control (0; red). (C and D) PHONEMeS solution model of signaling for mTOR phospho-antibody array after CELF4 silencing in QGP-1 (C) and BON-1 (D). Target proteins (purple circles) correspond to the highly regulated proteins, which were connected to its target phosphorylation sites (red circles) through intermediary kinases (blue circles). Central kinases, which were also identified by kinase activation prediction, are shown as intermediary kinases with small yellow circles. (E and F) Validation by Western blot assay of TSC2 and AKT phosphorylation in QGP-1 (E) and BON-1 (F) with representative images, normalized by TUBB.

Article Snippet: Cell lines transfected with siRNAs were lysed to analyze protein expression and phosphorylation by western blot, using standard procedures and specific antibodies for phospho-AKT (#4060S, Cell Signaling, Beverly, MA, USA); phospho-TSC2 (BS-3444R, Bioss, Woburn, MA, USA); and TUBB (#2128, Cell Signaling), as well as with the secondary antibody HRP-conjugated goat-anti-rabbit (#7074s; Cell Signaling) immunoglobulin G (IgG).

Techniques: Expressing, Functional Assay, Control, Ab Array, Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

doi: 10.1038/s41598-025-24699-w

Figure Lengend Snippet: Constitutive rapamycin sensitive activation of mTORC1 in MΦ TSC2−/− BMDMs favors pro-inflammatory polarization responses in vitro. A , Immunoblot of TSC2 protein expression in BMDMs from MΦ TSC2+/+ versus MΦ TSC2−/− mice ( n = 5 per group, unpaired t-test). B , Upper : Immunoblot for P70S6K and 4E-BP1 phosphorylation and total protein in BMDMs from same two models incubated with vehicle or rapamycin. ( n = 4 per group); Lower : phospho/total protein. C , Left : Immunoblot of Akt activation from same experiment ( n = 4 per group), Right : Summary data; all analyzed by one-way ANOVA, Dunnett multiple correction test. D , Proliferation of BMDMs from both groups with vehicle (Con), LPS/IFN-γ or IL-4 stimulation. Upper : cell numbers ( n = 20/group), Lower : cell size ( n = 60/group). E , Response of BMDMs isolated from each model to IFN-γ + LPS stimulation ± rapamycin ( n = 6/group), F BMDM responses to IL-4 ± rapamycin ( n = 6/group). Data and mean ± SEM displayed, data normalized to MΦ-TSC2 +/+ results for each analysis, using two-way ANOVA with Šidák multiple comparisons test. * P < 1e-20 vs. MΦTSC2 +/+ , † P < 1e-10, § P < 0.05 vs. vehicle within genotype. 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; P70S6K, ribosomal protein S6 kinase; Arg1, arginase 1; LPS, lipopolysaccharide; Mrc1, mannose receptor C-type 1; Nos2, nitric oxide synthase 2; Retnla, resistin-like alpha; TNFα, tumor necrosis factor alpha, other abbreviations in text.

Article Snippet: The I/R protocol was performed in three different cohorts: (1) control and MΦ TSC2−/− mice; (2) the same groups pre-administered 400 μg anti-Ly6G antibody (clone 1A8, BioXCell) i.p. starting 24 h prior to I/R and dosed 2x/week for 4 weeks (neutrophil depletion group) following established protocol ; and (3) control and MΦ TSC2−/− randomized to receive vehicle (3.3 mL/kg of 0.9% NaCl, 5% PEG400, 5% Tween 20, and 3% ethanol) or rapamycin (Target Mol Cat#53123-88-9, 2 mg/kg in vehicle) daily by intraperitoneal injection.

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Phospho-proteomics, Incubation, Isolation, Binding Assay

Journal: Scientific Reports

Article Title: Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

doi: 10.1038/s41598-025-24699-w

Figure Lengend Snippet: MΦTSC2 −/− mice are protected against post-I/R cardiac dysfunction. A , Representative echocardiographic images at baseline and 4 weeks post– I/R in MΦ TSC2+/+ and MΦ TSC2−/− mice. B , Quantification of left ventricular ejection fraction, end-diastolic short axis diameter, stroke diameter (end-diastolic – end-systolic diameter), and percent anterior wall thickening ( n = 6 per group). C , Lung wet-to-dry weight ratios ( n = 6 per group). D , Masson’s trichrome stained cross section with percent fibrosis area ( n = 5 per group, unpaired t-test). E , Gene expression for hypertrophic and matrix remodeling genes, data normalized to MΦ TSC2−/− (WT) Sham. A-type natriuretic peptide, Nppa , b-myosin heavy chain, Myh7 , collagen type 2a1, Col2a1 , metalloproteinase 2, Mmp2 ( n = 6/group). All data shown with mean ± SD, panels B , C , and E analyzed with two-way ANOVA with Šidák multiple comparisons test.

Article Snippet: The I/R protocol was performed in three different cohorts: (1) control and MΦ TSC2−/− mice; (2) the same groups pre-administered 400 μg anti-Ly6G antibody (clone 1A8, BioXCell) i.p. starting 24 h prior to I/R and dosed 2x/week for 4 weeks (neutrophil depletion group) following established protocol ; and (3) control and MΦ TSC2−/− randomized to receive vehicle (3.3 mL/kg of 0.9% NaCl, 5% PEG400, 5% Tween 20, and 3% ethanol) or rapamycin (Target Mol Cat#53123-88-9, 2 mg/kg in vehicle) daily by intraperitoneal injection.

Techniques: Staining, Gene Expression

Journal: Scientific Reports

Article Title: Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

doi: 10.1038/s41598-025-24699-w

Figure Lengend Snippet: Rapamycin prevents protection against cardiac I/R injury in MΦ TSC2–/– mice. A , B Example M-mode echocardiograms and summary data at 4 weeks post sham or I/R intervention in MΦ TSC2+/+ and MΦ TSC2–/– mice treated with either vehicle or rapamycin ( n = 5/group). Same parameters and analysis as shown in Fig. . C Lung wet-to-dry weight ratio, ( n = 5/group). D Representative heart sections and fibrotic area quantitation ( n = 7/group, unpaired t-test). E Gene expression of hypertrophy and matrix remodeling genes in same hearts ( n = 6/group). Analysis in panels B , C , and E are by two-way ANOVA with Šidák multiple comparison test. All data are shown along with mean ± SD.

Article Snippet: The I/R protocol was performed in three different cohorts: (1) control and MΦ TSC2−/− mice; (2) the same groups pre-administered 400 μg anti-Ly6G antibody (clone 1A8, BioXCell) i.p. starting 24 h prior to I/R and dosed 2x/week for 4 weeks (neutrophil depletion group) following established protocol ; and (3) control and MΦ TSC2−/− randomized to receive vehicle (3.3 mL/kg of 0.9% NaCl, 5% PEG400, 5% Tween 20, and 3% ethanol) or rapamycin (Target Mol Cat#53123-88-9, 2 mg/kg in vehicle) daily by intraperitoneal injection.

Techniques: Quantitation Assay, Gene Expression, Comparison

Journal: Scientific Reports

Article Title: Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

doi: 10.1038/s41598-025-24699-w

Figure Lengend Snippet: Reduced myocardial pro-inflammatory immune cell infiltration 5 days post-I/R in MΦ TSC2−/− mice. A Number of CD45 + per mg tissue between MΦTSC2 +/+ and MΦTSC2 –/– hearts ( n = 5/group). B Number of CD64 + macrophages per mg in hearts from both models ( n = 5/group). C , Representative flow cytometry density plots for CCR2 + /CD64 + macrophages ( n = 5/group). D , Number of Ly6C + monocytes per mg tissue in both groups, ( n = 5/group). E Representative flow cytometry analysis of macrophage subsets ( n = 5/group). F Ly6G + (neutrophils), CD8 + and CD4 + (T cells), and CD19 + (B cells) each normalized to mg tissue for both groups ( n = 5). Analysis used two-tailed unpaired t-tests. Data are mean ± SEM. CD45, leukocyte common antigen; CCR2, C-C chemokine receptor type 2; CD64, Fc gamma receptor 1; Ly6C, lymphocyte antigen 6 complex locus C; Ly6G, lymphocyte antigen 6 complex locus G; MHCII, major histocompatibility complex class II; SEM, standard error of the mean; TSC2, tuberous sclerosis complex 2.

Article Snippet: The I/R protocol was performed in three different cohorts: (1) control and MΦ TSC2−/− mice; (2) the same groups pre-administered 400 μg anti-Ly6G antibody (clone 1A8, BioXCell) i.p. starting 24 h prior to I/R and dosed 2x/week for 4 weeks (neutrophil depletion group) following established protocol ; and (3) control and MΦ TSC2−/− randomized to receive vehicle (3.3 mL/kg of 0.9% NaCl, 5% PEG400, 5% Tween 20, and 3% ethanol) or rapamycin (Target Mol Cat#53123-88-9, 2 mg/kg in vehicle) daily by intraperitoneal injection.

Techniques: Flow Cytometry, Two Tailed Test, Immunopeptidomics

Journal: Scientific Reports

Article Title: Macrophages lacking TSC2 have mTORC1-dependent increased GPNMB and ameliorate ventricular dysfunction/remodeling after ischemia-reperfusion

doi: 10.1038/s41598-025-24699-w

Figure Lengend Snippet: Enhanced GPNMB protein in MΦ TSC2–/– BMDM and post IR-myocardium. A Western blot and quantification show increased GPNMB protein levels in BMDMs isolated from MΦ TSC2–/– mice compared to MΦ TSC2+/+ controls measured under basal conditions ( n = 5 per group, unpaired two-tailed t-test). B GPNMB levels elevated in MΦ TSC2–/– BMDMs are significantly reduced after rapamycin treatment ( n = 4 per group). C Western blot of cardiac tissue lysates 4 weeks post–I/R shows increased GPNMB expression in MΦ TSC2–/– hearts that is absent in the cohort treated with rapamycin ( n = 4 per group, D ELISA quantification of GPNMB protein from the same cardiac tissues confirmed the elevation in MΦTSC2 –/– hearts and its suppression by rapamycin ( n = 6 per group). Analysis in panels B – D , one-way ANOVA with Dunnett multiple comparisons correction. Data are mean ± SD. Rapa – rapamycin.

Article Snippet: The I/R protocol was performed in three different cohorts: (1) control and MΦ TSC2−/− mice; (2) the same groups pre-administered 400 μg anti-Ly6G antibody (clone 1A8, BioXCell) i.p. starting 24 h prior to I/R and dosed 2x/week for 4 weeks (neutrophil depletion group) following established protocol ; and (3) control and MΦ TSC2−/− randomized to receive vehicle (3.3 mL/kg of 0.9% NaCl, 5% PEG400, 5% Tween 20, and 3% ethanol) or rapamycin (Target Mol Cat#53123-88-9, 2 mg/kg in vehicle) daily by intraperitoneal injection.

Techniques: Western Blot, Isolation, Two Tailed Test, Expressing, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: CD168 + macrophages promote hepatocellular carcinoma tumor stemness and progression through TOP2A /β-catenin/ YAP1 axis

doi: 10.1016/j.isci.2023.106862

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-Ki-67 , Boster , Cat#M00254-9.

Techniques: Recombinant, Preserving, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: Whole egg, but not egg white, ingestion induces mTOR colocalization with the lysosome after resistance exercise

doi: 10.1152/ajpcell.00225.2018

Figure Lengend Snippet: Tuberous sclerosis 2 (TSC2) and mechanistic target of rapamycin (mTOR) colocalization with Ras homolog enriched in brain (Rheb) in response to whole eggs or egg whites following resistance exercise. Immunofluorescence quantification of mTOR/TSC2 (red) and Rheb (green) colocalization, displayed as a composite image (merge) and WGA (blue) (n = 10/condition). Yellow/orange regions represent TSC2 and Rheb (A) mTOR and Rheb colocalization (C). Each panel represents one subject from whole eggs and egg whites across the experimental time course. Group data are quantified and reported as TSC2 and Rheb colocalization (B) and mTOR and Rheb colocalization (D). Open circles represent whole eggs and closed circles represent egg whites. All data are presented relative to Fasted. Scale bar represents 100-μm area. Data are presented as means ± SE and were analyzed using two-way repeated-measures ANOVA. *P < 0.01, different from Fasted in same condition. Immunofluorescence image was obtained on a Leica DMI8 confocal microscope of mTOR (red) and Rheb (green) interaction, displayed as a composite image (merge) and WGA (blue) (E). Sections were viewed at ×60 1.30 numerical aperture oil immersion objective to visualize the signal being primarily intracellular and/or within the fiber junctions. Scale bar represents 20-μm area (E).

Article Snippet: The mouse monoclonal anti-mTOR (no. 05-1592) antibody was purchased from Millipore (Toronto, ON, Canada) and mouse monoclonal anti-TSC2 (no. AM1919b-EV) was purchased from FroggaBio (Toronto, ON, Canada).

Techniques: Immunofluorescence, Microscopy