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

Journal: Toxins

Article Title: Autophagy and Apoptosis Interact to Modulate T-2 Toxin-Induced Toxicity in Liver Cells

doi: 10.3390/toxins11010045

Figure Lengend Snippet: Interactions of the autophagic and apoptotic effects of L02 cells induced by T-2 toxin. ( A ) Effect of RAPA on autophagy caused by T-2 toxin. L02 cells were pre-exposed to the autophagy stimulator RAPA (100 nM) for 24 h, and then co-treated with T-2 toxin (5 nM) for an additional 12 h. The levels of p62 and Beclin 1 proteins and the LC3-II/LC3-I ratio were analyzed by Western blotting (WB). ( B ) Effect of RAPA on apoptosis caused by T-2 toxin. L02 cells were pretreated with the autophagy stimulator RAPA (100 nM) for 24 h, and then co-treated with T-2 toxin (5 nM) for an additional 12 h. The levels of the apoptosis-related proteins of PARP-1 and caspase-3 as well as the Bax/Bcl-2 ratio were analyzed by Western blotting (WB). ( C ) Effect of the autophagy inhibitor CQ on autophagy- and apoptosis-associated proteins in L02 cells exposed to T-2 toxin. L02 cells were pretreated with the autophagy inhibitor CQ (100 μM) for 1 h, and exposure to the inhibitor was continued during subsequent T-2 toxin (5 nM) treatment for 6 h. The LC3-II/LC3-I ratio, Beclin 1 level, and caspase-3 proteins were analyzed by Western blotting. ( D ) Effect of CQ on T-2 toxin-induced apoptosis. L02 cells were pretreated with the autophagy inhibitor CQ (100 μM) for 1 h, and then co-treated with T-2 toxin (5 nM) for an additional 6 h. Western blotting was used for the analysis of caspase-3 proteins. ( E ) The autophagic rate caused by T-2 toxin was greatly enhanced upon the inhibition of autophagy. The L02 cells were treated as described in ( D ) and cell apoptosis was detected using flow cytometry. Results are the mean ± SD, n = 3. ** represents p < 0.01 and * represents p < 0.05.

Article Snippet: Rapamycin (RAPA) and chloroquine diphosphate salt (CQ) were obtained from Solarbio (Beijing, China). pGMLV-CMV-RFP-GFP-hLC3 lentivirus was purchased from Genomeditech (Shanghai, China).

Techniques: Western Blot, Inhibition, Flow Cytometry

Journal: Neuroscience Bulletin

Article Title: Increased EZH2 Levels in Anterior Cingulate Cortex Microglia Aggravate Neuropathic Pain by Inhibiting Autophagy Following Brachial Plexus Avulsion in Rats

doi: 10.1007/s12264-020-00502-w

Figure Lengend Snippet: Upregulation of autophagy suppresses neuroinflammation and attenuates neuropathic pain. A, B The 50%MWT (A) and CWS (B) in the sham and neuropathic pain groups after rapamycin treatment from day 3 to day 14 post-surgery (mean ± SD; n = 8; n.s., no significant difference among Sham, NeuP and GSK126 groups; *P < 0.05, **P < 0.01 vs NeuP group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs Sham group). C, D Relative expression of LC3II (C) and p62 protein (D) in the ACC of the Sham and NeuP groups after rapamycin treatment compared with DMSO treatment from day 3 to day 14 post-surgery. E–G ELISA analysis of the IL-1β (E), TNF-α (F), and IL-6 (G) expression levels in the ACC of the Sham and NeuP groups after rapamycin treatment from day 3 to day 14 post-surgery. For C–G: mean ± SD; n = 8; n.s., no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, vs DMSO group.

Article Snippet: Intraperitoneal Drug Administration GSK126, 3-methyladenine (3-MA), and rapamycin (Rapa) from APExBIO Technology (Houston, TX, USA) were dissolved in PBS with 5% dimethyl sulfoxide (DMSO).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Baicalin ameliorates lupus autoimmunity by inhibiting differentiation of Tfh cells and inducing expansion of Tfr cells

doi: 10.1038/s41419-019-1315-9

Figure Lengend Snippet: a Naive T cells sorted from MRL/lpr mice were cultured in the presence of IL-21 and IL-6 with or without 10 μM mTOR agonist MH1485, 200 ng/ml mTOR antagonist rapamycin (RAPA) or 40 μM Baicalin for 5 days. CXCR5 + PD-1 + cells were analyzed by flow cytometry using the CD4 + gate (left). The results of flow cytometry of CD4 + CXCR5 + PD-1 + cells (right). Results shown are representative of three biological independent experiments. b Naive T cells from MRL/lpr mice were cultured in the presence of TGF-β and IL-2 with or without 10 μM MH1485, 200 ng/ml RAPA or 40 μM Baicalin for 5 days. CXCR5 + Foxp3 + cells were analyzed by flow cytometry using the CD4 + gate (left). The results of flow cytometry of CD4 + CXCR5 + Foxp3 + cells (right). Results shown are representative of three biological independent experiments. c Sorted naive T cells were cultured with TGF-β, IL-2 with or without 40 μM Baicalin or 10 μM MHY1485 for 3 h, P-mTOR, p-S6K, P-4EBP1, and GAPDH expression were analyzed by western blot. d Twelve-week-old of MRL/lpr mice were treated intraperitoneally with 200 mg/kg Baicalin or PBS vehicle daily for 4 weeks. P-mTOR and GAPDH expression in spleen were analyzed by western blot. MRL/lpr mice were treated intraperitoneally with 1.5 mg/kg/d RAPA with or without 200 mg/kg of Baicalin daily for 4 weeks, e RAPA and Baicalin treatment inhibited spleen enlargement and reduced the spleen index ( n = 4 for each group). f RAPA and Baicalin treatment reduced the percentage of CD4 + CXCR5 + PD-1 + Tfh cells in the spleens of MRL/lpr mice ( n = 4 for each group). g RAPA and Baicalin treatment promoted the percentage of CD4 + CXCR5 + Foxp3 + cells in the spleens of MRL/lpr mice ( n = 4 for each group). *, p < 0.05. ANOVA and Student’s t -test were used

Article Snippet: For some experiments, MRL/lpr mice were treated intraperitoneally with 1.5 mg/kg/d mTOR antagonist rapamycin (RAPA, LC Laboratories) with or without 200 mg/kg of Baicalin daily for 4 weeks.

Techniques: Cell Culture, Flow Cytometry, Expressing, Western Blot

Journal: Mediators of Inflammation

Article Title: mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections

doi: 10.1155/2016/7369351

Figure Lengend Snippet: E. coli infection activated mTORC1/S6k pathway in monocytes, and TSC1 KO mice partly simulated E. coli infection. (a) The percentage of EYFP in monocytes from Rosa-LysMCreTsc1 flox/flox mice. This percentage represents the TSC1 deletion ratio in TSC1 KO monocytes. (b) The phosphorylation level of S6k in WT and TSC1 KO monocytes (CD11b + Ly6C + Ly6G − ) in the presence or absence of Rapa (1.5 mg/kg, four days before treatment) (MFI: median fluorescence intensity) (three mice per group, three representative experiments). (c) The phosphorylation level of S6k in WT and TSC1 KO monocytes (CD11b + Ly6C + Ly6G − ) with or without E. coli infection (5 ∗ 10 7 cells/mouse) for 8 h (three mice per group, three representative experiments). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 compared to WT mice or between the indicated groups. P values were determined using Student's t -tests.

Article Snippet: Rapamycin (Rapa) (Cayman Chemical, Country) was dissolved in PBS containing 5% DMSO.

Techniques: Infection, Phospho-proteomics, Fluorescence

Journal: Mediators of Inflammation

Article Title: mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections

doi: 10.1155/2016/7369351

Figure Lengend Snippet: Change in cytokine expression by monocytes in mice with infected bacteria. Freshly isolated peritoneal monocytes (CD11b + Ly6G − Ly6C + ) were sorted from WT, TSC1 KO, and TSC1 + Rapa (1.5 mg/kg, four days before treatment) mice after E. coli infection (5 ∗ 10 7 cells/mouse), and the expression of TNF- α , IFN- γ , IL-1 β , IL-10, and TGF- β was determined by real-time PCR ( n = 3 representative experiments). ∗ P < 0.05 and ∗∗ P < 0.01 compared to WT mice or between the indicated groups. P values were determined using Student's t -tests.

Article Snippet: Rapamycin (Rapa) (Cayman Chemical, Country) was dissolved in PBS containing 5% DMSO.

Techniques: Expressing, Infection, Bacteria, Isolation, Real-time Polymerase Chain Reaction

Journal: Mediators of Inflammation

Article Title: mTORC1-Activated Monocytes Increase Tregs and Inhibit the Immune Response to Bacterial Infections

doi: 10.1155/2016/7369351

Figure Lengend Snippet: ROS production in monocytes. There was significantly higher ROS production in monocytes from TSC1 KO mice (CD11b + Ly6c + Ly6G − ) than from WT mice. The difference was attenuated in monocytes with the Rapa treatment (1.5 mg/kg, four days before treatment) (four mice per group, three representative experiments). ∗ P < 0.05 compared to WT mice or between the indicated groups. P values were determined using Student's t -tests.

Article Snippet: Rapamycin (Rapa) (Cayman Chemical, Country) was dissolved in PBS containing 5% DMSO.

Techniques:

Journal: Oncology Research

Article Title: Trametinib boosts palbociclib’s efficacy in breast cancer via autophagy inhibition

doi: 10.32604/or.2024.046139

Figure Lengend Snippet: TRA inhibits PAL-induced autophagy in breast cancer. (A) Representative images depicting GFP-LC3 puncta in U87 cells treated with PAL in the presence or absence of TRA at indicated concentrations. Magnification: 20×. Scale bar: 100 μm. (B) A bar chart showing the average number of GFP-LC3 puncta per cell. (C) Western blotting analysis of LC3 from the protein lysate of PAL-treated MCF7 cells with or without TRA. (D) A bar chart indicating the ratio of LC3-II to LC3-I. (E) Western blotting analysis of LC3 from the protein lysate of tumor tissue extracted from nude mice treated with PAL, with or without TRA. (F) A bar chart showing the ratio of LC3-II to LC3-I in the tumor tissue. (G) Representative immunohistochemical analysis images of LC3 in tumor tissue from PAL-treated nude mice, with or without TRA. Magnification: 40×. Scale bar: 50 μm. (H) A bar chart indicating the percentage of cells positive for LC3 in the tumor tissue. Bar. S.D., *** p < 0.001. Full unedited gel/blots are provided in Suppl. Fig. S4, where the protein molecular weight markers were labeled.

Article Snippet: TRA, rapamycin (RAPA), bafilomycin A1 (Baf), and PAL were obtained from Topscience Co. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) to create stock solutions at appropriate concentrations for in vitro assays.

Techniques: Western Blot, Immunohistochemical staining, Molecular Weight, Labeling

Journal: Oncology Research

Article Title: Trametinib boosts palbociclib’s efficacy in breast cancer via autophagy inhibition

doi: 10.32604/or.2024.046139

Figure Lengend Snippet: TRA augments PAL sensitivity in breast cancer. (A, B) Bar charts showing the cell viability of PAL-treated MCF7 and MDA-MB-468 cells with or without TRA at indicated concentrations for 24 h. (C) Representative images of cellular morphology images for PAL-treated MCF7 cells and MDA-MB-468 cells with or without TRA at indicated concentrations for 24 h. Magnification: 10×. Scale bar: 200 μm. (D) Representative crystal violet staining images displaying the colony formation of PAL-treated MCF7 cells and MDA-MB-468 cells with or without TRA at indicated concentrations. (E) Images showing the appearance of the MCF7 tumors extracted from nude mice treated with PAL, with or without TRA. (F) A curve line graph indicating the tumor volume of PAL-treated nude mice with or without TRA. (G) A curve line graph showing the body weight of PAL-treated nude mice with or without TRA. (H) Representative H&E staining images of tumor tissue from PAL-treated nude mice with or without TRA. Magnification: 10× (upper panel) and 20× (lower panel). Scale bar: 200 μm (upper panel) and 100 μm (lower panel). (I) Representative immunohistochemical analysis images of Ki67 in tumor tissue from PAL-treated nude mice with or without TRA. Magnification: 40×. Scale bar: 50 μm. (J) A bar chart indicating the percentage of cells positive for Ki67. Bar. S.D., * p < 0.05, ** p < 0.01, *** p < 0.001, ### p < 0.001 vs . PAL (100 mg/kg).

Article Snippet: TRA, rapamycin (RAPA), bafilomycin A1 (Baf), and PAL were obtained from Topscience Co. (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) to create stock solutions at appropriate concentrations for in vitro assays.

Techniques: Staining, Immunohistochemical staining