Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A ) Changes in in-cell LiP-MS peptide intensities in HEK293 cells treated with 20 μM rapamycin compared to DMSO control. Each data point represents a single peptide; half-tryptic peptides of FKBP1A are shown in orange, fully-tryptic peptides of FKBP1A are shown in blue. The lines marks significance levels (FC > 1.5, two-sample unpaired t-test, p -value < 0.01, n = 6 technical replicates). ( B ) Changes in standard LiP-MS peptide intensities in a native lysate of HEK293 cells treated with 10 nM rapamycin compared to DMSO control (6 technical replicates). Each data point represents a single peptide; half-tryptic peptides of FKBP1A are shown in orange, fully-tryptic peptides of FKBP1A are shown in blue. The shaded gray region marks significance levels (FC > 1.5, two-sample unpaired t-test, p -value < 0.01, n = 4 technical replicates). The four FKBP1A peptides with the lowest p -value are highlighted. ( C ) Overall sequence coverage of experiments in ( A , B ). The plot shows the indicated quantities across both conditions (in-cell LiP-MS n = 12 technical replicates; standard LiP-MS n = 8 technical replicates). Error bars are shown in black. ( D ) Number of peptides with missing values per treatment in ( A , B ). The plots report the number of replicates per condition, in which a specific peptide was not quantified. A missing value of 0 indicates that the peptide was quantified in all six replicates, 1 indicates that the peptide was not quantified in 1 out of 6 replicates, and so on. ( E ) Coefficient of variation (CV) of peptide intensities per treatment in ( A , B ). ( F ) Fraction of half-tryptic peptides relative to total peptide intensity for the experiments in ( A , B ). ( G , H ) Plots show the indicated quantities across both conditions (in-cell LiP-MS n = 12; standard LiP-MS n = 8). ( G ) Fraction of peptides with missed cleavages after tryptic digestion relative to total peptide intensity for the experiments in ( A , B ). ( H ) Fraction of peptides with indicated length relative to total peptide intensity in ( A , B ). Only peptides with up to 50 amino acids are shown. To generate technical replicates, cells were split into the indicated number of aliquots before treatment.

Article Snippet: For the analysis of organelle and domain coverage, cells were treated with 0.016% DMSO for 5 min prior to in-cell LiP-MS. For quantitative immunoblots, HEK293T cells seeded in 12-well plates were treated with 100 nM or 250 nM Rapamycin (Thermo Fisher, J62473) at 70–80% confluency for indicated time points in normal growth medium.

Techniques: Control, Sequencing

Journal: Molecular Systems Biology

Article Title: Limited proteolysis-coupled mass spectrometry captures proteome-wide protein structural alterations and biomolecular condensation in living cells

doi: 10.1038/s44320-025-00182-6

Figure Lengend Snippet: ( A – E ) In-cell LiP-MS of HEK293 cells treated with 20 μM rapamycin compared to DMSO control; standard LiP-MS in a native lysate of HEK293 cells treated with 10 nM rapamycin compared to DMSO control. Plots ( B – E ) show indicated quantities across both conditions (in-cell LiP-MS n = 12; standard LiP-MS n = 8 technical replicates). Error bars are shown in black. ( A ) Heatmap of peptide intensities (stripped sequence level). Colors above the heatmap correspond to the indicated conditions. ( B ) Number of detected proteins. ( C ) Number of detected peptides. ( D ) Overall sequence coverage considering peptides in both rapamycin treated and control samples (in-cell LiP-MS n = 12 replicates; standard LiP-MS n = 8 technical replicates). ( E ) Number of peptides with indicated length relative to total peptide intensity. Only peptides with up to 50 amino acids are shown. To generate technical replicates, cells were split into the indicated number of aliquots before treatment. ( F ) HEK293T cells were treated with rapamycin under the indicated conditions and probed for mTORC1 or mTORC2 activity using Western blots against the indicated phospho-proteins. The plots (right) show quantification of the Western blots on the left. Statistics were performed with two-way ANOVA on three biological replicates (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001). Each replicate was biologically independent, consisting of cells grown separately prior to treatment.

Article Snippet: For the analysis of organelle and domain coverage, cells were treated with 0.016% DMSO for 5 min prior to in-cell LiP-MS. For quantitative immunoblots, HEK293T cells seeded in 12-well plates were treated with 100 nM or 250 nM Rapamycin (Thermo Fisher, J62473) at 70–80% confluency for indicated time points in normal growth medium.

Techniques: Control, Sequencing, Activity Assay, Western Blot

Journal: Frontiers in Behavioral Neuroscience

Article Title: Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics

doi: 10.3389/fnbeh.2015.00062

Figure Lengend Snippet: mTOR signaling pathway is required for neurite elongation induced by 5-HT7R stimulation . Cortical neurons were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), or the mTOR inhibitors rapamycin (RAPA, 20 nM), or torin 1 (TR1, 250 nM) with or without LP. (A) Representative Tuj1 immunostaining of neuronal cultures (magnification 20x). The images in the lower row are the same of the upper row with the addition of the dashed yellow lines manually drawn by the operator from the soma (yellow circle) to the end of the primary neurite in order to measure neurite length. (B) The graph shows the neurite length expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The bars represent means ± SEM from randomly selected fields for each cell culture condition ( n = 9). Asterisk (*): value significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test ( p < 0.05).

Article Snippet: The mTOR inhibitors rapamycin (Sigma-Aldrich) and torin 1 (Tocris), were used at a final concentration of 20 and 250 nM, respectively.

Techniques: Immunostaining, Cell Culture

Journal: Frontiers in Behavioral Neuroscience

Article Title: Activation of 5-HT7 receptor stimulates neurite elongation through mTOR, Cdc42 and actin filaments dynamics

doi: 10.3389/fnbeh.2015.00062

Figure Lengend Snippet: Stimulation of 5-HT7R activates mTORC1 signaling . Cortical neurons were treated for 2 h with the 5-HT7R selective agonist LP-211 (LP, 100 nM), or the mTOR inhibitor torin 1 (TR1, 250 nM), alone or in combination. (A) The bars show the level of p70S6K phosphorylation at Thr389 (means ± SEM, n = 6), expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The level of p70S6K phosphorylation was measured by Western blot as intensity of p70S6K phosphorylated at Thr389 (p-p70S6K) normalized with that of total (phosphorylated and unphosphorylated) p70S6K in the same samples. The inset displays representative blots probed with antibodies against p-p70S6K and p70S6K; the molecular weights (kDa) are shown on the right. (B) The bars show the level of Akt phosphorylation at Ser473 (means± SEM, n = 3), expressed as percentage of values measured in the corresponding vehicle-treated cultures (CTRL, set to 100%). The level of Akt phosphorylation was measured by Western blot as intensity of Akt phosphorylated at Ser473 (p-Akt) normalized with that of total Akt (Akt) in the same samples. The inset displays representative blots probed with antibodies against p-Akt and Akt; the molecular weights (kDa) are shown on the right. Asterisk (*): value significantly different from CTRL by One Way ANOVA followed by Dunnett post-hoc test ( p < 0.05).

Article Snippet: The mTOR inhibitors rapamycin (Sigma-Aldrich) and torin 1 (Tocris), were used at a final concentration of 20 and 250 nM, respectively.

Techniques: Phospho-proteomics, Western Blot

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 1 Intracellular concentrations of TAC in human islets. Human islets were cultured with TAC (10 or 30 lg/l), SRL (10 or 30 lg/l), or the combi- nation thereof for 24–48 h before the intracellular concentration of TAC was measured in islet lysate and normalized to total protein as detailed in methods. Data are presented as the mean SD, n = 6 for each group. TAC, tacrolimus; SRL, sirolimus; *P < 0.04; ** P < 0.007; *** P < 0.0006.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture, Concentration Assay

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 2 Intracellular concentrations of SRL in human islets. Human islets were cultured with TAC (10 or 30 lg/l), SRL (10 or 30 lg/l), or the combi- nation thereof for 24–48 h before the intracellular concentration of SRL was measured in islet lysate and normalized to total protein as detailed in methods. Data are presented as the mean SD, n = 6 for each group. TAC: tacrolimus; SRL: sirolimus; *** P < 0.001; **** P < 0.0001.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture, Concentration Assay

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 3 Effect of CsA on intracellular concentration of SRL in human islets. Human islets were cultured with the combination of SRL (30 lg/l) and CsA (5 lg/ml), or the drug alone for 24 h before the intracellular concentration of SRL (a) or CsA (b) was measured in islet lysate and nor- malized to total protein as detailed in methods. Data are calculated as percentages of control and are presented as mean SD, n = 6 for each group. CsA, cyclosporine A; SRL, sirolimus.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Concentration Assay, Cell Culture, Control

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 4 The effect of SRL, TAC, or CsA on phosphorylation of p70S6k in islets. Human islets were cultured with TAC (30 lg/l), SRL (30 lg/l), or the combination thereof for 24 h before the presence of p- p70s6k was assessed by the cell-signaling Bio-Plex assay in human islet cell lysate and normalized to total protein (a). In a parallel experiment, human islets were cultured with SRL (30 lg/l), CsA (5 lg/ml), or the combination thereof for 24 h before p-p70S6k was detected in the lysate and normalized to total protein. Data are calculated as ratio to control and are presented as mean SD, n = 3–6 for each group. TAC, tacrolimus; SRL, sirolimus; CsA, cyclosporine A, ** P < 0.01, **** P < 0.0001.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Phospho-proteomics, Cell Culture, Plex Assay, Control

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 5 Oxygen consumption rates (OCR) in human islets after treatment of TAC, SIR, or SIR+TAC. Human islets were cultured with TAC (30 lg/l), SIR (30 lg/l), or a combination thereof for 24 h before the glucose-stimulated OCR was measured as indicated in methods. OCR is expressed as percentage of baseline and is presented as the mean SD, n = 6 for each group. TAC, tacrolimus; SRL, sirolimus, ** P < 0.01.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Cell Culture

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Intracellular sirolimus concentration is reduced by tacrolimus in human pancreatic islets in vitro.

doi: 10.1111/tri.12617

Figure Lengend Snippet: Figure 6 Expression of ABCB1 (Pgp), OATP1B1, and CYP3A4 in human islets. Human islets were cultured for 24 h before the expression of the drug transporter (ABCB1(Pgp) and OATP1B1), and the metabolic enzyme CYP3A4 was evaluated. RNA was prepared and subjected to qPCR as detailed in methods. The reference gene index is calculated by the mean of ALAS1, B2M, and RPL13A expression and used to normalize the expression of target genes in isolated hepatocytes relative to the mRNA level of ABCB1(Pgp), OATP1B1, and CYP3A4 in human islets (a). OATB1 mRNA expression in human islets was normalized to reference gene index after exposure to TAC (30 lg/l), SRL (30 lg/l), or a combination thereof for 24 h (b). Represen- tative immunofluorescence image of dispersed human islets stained for insulin (green), ABCB1(Pgp) (red) and nuclear staining with DAPI (blue) (c), or glucagon (green), ABCB1(Pgp) (red) and nuclear staining wit DAPI (blue) (d). Data are presented as mean SD, n = 4–5 for each group. TAC, tacroli- mus; SRL, sirolimus; *P < 0.05; ** P < 0.01.

Article Snippet: The islets were exposed to 10 and 30 lg/l of tacrolimus (Santa Cruz Biotechnology, Dallas, TX, USA) or sirolimus (Toronto Research Chemicals, Toronto, Ontario, Canada) or the combination thereof for 24 h and 48 h at 37 °C (5% CO2).

Techniques: Expressing, Cell Culture, Isolation, Staining