Journal: Journal of Cellular and Molecular Medicine

Article Title: Dual inhibition of BDNF/TrkB and autophagy: a promising therapeutic approach for colorectal cancer

doi: 10.1111/jcmm.13181

Figure Lengend Snippet: Effect of tyrosine kinase receptor inhibitor (K252a) on TrkB/BDNF expression and autophagy induction. SW480 and SW620 were treated with 100 nM of K252a for 3 hrs, recovered, washed and total RNA and proteins were extracted (see ). ( A ) BDNF, Full Length (FL) and Truncated (Tr) TrkB, transcripts expression was assessed by qPCR. Histograms are representative of three independent experiments. The fold expression was obtained by the comparative cycle threshold method using the GAPDH RNA expression level as internal control. ( B ) BDNF, FL TrkB, Tr TrkB, phospho‐TrkB (P TrkB), AKT, phospho‐AKT (P AKT), mTOR and phospho‐mTOR (P mTOR), proteins expression were assessed by Western blotting. The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (* P < 0.05; ** P < 0.01; *** P < 0.001). ( C ) Staining of SW480 and SW620 was realized with anti‐BDNF antibody (green), anti‐TrkB antibody (red) and DAPI (blue). Images were obtained through confocal microscopy (magnification ×1000). ( D ) Beclin1, ATG5 and LC3 proteins expression was assessed by Western blotting after treatment with either K252a (100 nM, 3 hrs) or with CQ (25 μM, 3 hrs). The density of each band representative for protein expression was calculated with ImageJ software. Images show representative results of three experiments. Statistically results are explained in comparison with control cells, normalized at 1 (* P < 0.05; ** P < 0.01; *** P < 0.001). ( E ) Staining of SW480 and SW620 was realized with anti‐LC3 antibody (green) and DAPI (blue) through indirect immunofluorescence. Rapamycin (20 nM, 3 hrs) was used as a positive control for autophagy induction. Relative fluorescence quantification was accomplished by using a green surface plot with the ImageJ software application.

Article Snippet: K252a (Alomone Labs, Jerusalem, Israel), chloroquine (CQ) and rapamycin (Sigma‐Aldrich, St. Quentin Fallavier, France) were used for NT, autophagy and mTOR inhibition, respectively.

Techniques: Expressing, RNA Expression, Western Blot, Software, Staining, Confocal Microscopy, Immunofluorescence, Positive Control, Fluorescence

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: American Journal of Translational Research

Article Title: Astragaloside IV prevents myocardial hypertrophy induced by mechanical stress by activating autophagy and reducing inflammation

doi:

Figure Lengend Snippet: Evidence that astragaloside IV treatment can improve cardiac function and reduce the myocardial hypertrophy induced by AB at 6 weeks. (A) Left ventricular ejection fraction. (B) Hypertrophy indexes. (C) Heart weight index (HWI = heart weight/body weight) and left ventricular weight index (LVWI = left ventricular weight/body weight) measurements. (D and E) Expression of hypertrophic marker proteins ANP and BNP as detected by Western blot. (F) HE staining. Sham, sham operation group; AB, aortic banding group; As-IV40, 40 mg/kg astragaloside IV-treated aortic banding group; As-IV80, 80 mg/kg astragaloside IV-treated aortic banding group; Rapa, rapamycin-treated aortic banding (positive control) group. Values are presented as the means ± SD, n = 4 for (A and C), n = 5 for (C), n = 3 for (D-F). #P < 0.05 compared with the sham operation group; *P < 0.05 compared with the model group.

Article Snippet: Rapamycin (Rapa) was purchased from Solarbio Co., Ltd. (Beijing, China).

Techniques: Expressing, Marker, Western Blot, Staining, Positive Control

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) is required for ERC‐based therapy in prolongation of cardiac allograft survival. (A): Survival time of cardiac allografts in each B6 recipients receiving different immunosuppressive treatments. Data were shown as percentage of cardiac allograft survival. Statistical analysis was done by Log‐rank (Mantel‐Cox) test. p < .001 (ERCs vs. *ERCs: 19.67 ± 2.58 days vs. 10.17 ± 1.17 days), p < .001, (ERCs + RAPA vs. *ERCs + RAPA: 100 days vs. 28.17 ± 1.72 days). (B): Histology of cardiac allografts in each group of B6 recipients. Grafts were collected at the time of rejection or POD 100, fixed by formalin, and embedded by paraffin. Sections (5 μm) were stained with H&E staining. Cardiac grafts from untreated (Ba) , RAPA monotherapy (Bb) , ERC monotherapy (Bc) , *ERCs (Bd) , ERCs‐RAPA combination group (Be) , or combination of *ERCs and RAPA (Bf) groups are presented and compared, n = 6. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Arrows indicate intravascular and/or interstitial changes in cardiac grafts (×400 magnification). Scale bars = 100 μm. Abbreviations: ERC, endometrial regenerative cell; POD, postoperative day; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Staining, Inhibition

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) mediates the role of ERC‐based therapy in reducing antibody‐mediated rejection in cardiac allografts. (A): Immunohistological staining of intragraft IgG ( Aa–Af ) and IgM ( Ag–Al ) antibody deposition of each group. Grafts were collected at the time of rejection or postoperative day (POD) 100. Arrows show positive staining (×400 magnification). (B): Intragraft IgG and IgM antibody deposition of each group were presented by the percentage of positive staining within a given section (mm 2 ). Grafts were collected at the time of rejection or POD 100. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Statistical analysis was done by one‐way analysis of variance followed by the least significant difference test, n = 6. Scale bars = 100 μm. Abbreviations: ERC, endometrial regenerative cell; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Staining, Inhibition

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) mediates the role of ERC‐based therapy in reducing acute cellular rejection in cardiac allografts . (A): Immunohistological staining of CD4 + (Aa–Af) and CD8 + (Ag–Al) cells infiltration of each group. Grafts were collected at the time of rejection or POD 100. Arrows show positive staining (×400 magnification). (B): Intragraft CD4 + and CD8 + cell infiltration of each group was presented by quantitating all the positive staining cells within a given section (cells per mm 2 ). Grafts were collected at the time of rejection or POD 100. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Statistical analysis was done by one‐way analysis of variance followed by the least significant difference test, n = 6. Scale bars = 100 μm. Abbreviations: ERC, endometrial regenerative cell; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Staining, Inhibition

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) mediates the effect of ERC‐based therapy in increasing the percentage of tolerogenic dendritic cell (Tol‐DCs) in transplant recipients. Splenocytes were harvested from B6 recipients at postoperative day 8, followed by double‐staining gated by anti‐mouse CD11c antibody, and then the percentage of surface MHC class II (A) , CD86 (B) , and CD40 (C) were measured by fluorescence‐activated cell sorting (FACS) analysis. Statistical analysis was done by one‐way analysis of variance (ANOVA) followed by the least significant difference (LSD) test, n = 6. (D): CD11c + DCs were isolated by CD11c microBeads from splenocytes collected from the B6 recipients and were treated with mitomycin. The function of these DCs (stimulators) was measured by the stimulation of CD3 + T‐cell (responders) proliferation index (OD value) in one‐way mixed lymphocyte reaction. Statistical analysis was done by one‐way ANOVA followed by the LSD test, n = 6. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Abbreviations: ERC, endometrial regenerative cell; OD, optical density; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Double Staining, Fluorescence, FACS, Isolation, Inhibition

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) mediates ERC‐based therapy in decreasing the percentage of total macrophages and but increasing the percentage of macrophage type 2 (M2) in transplant recipients. Splenocytes were collected from the B6 recipients in each group at postoperative day 8, followed by single‐staining with anti‐mouse CD68 antibody to measure the percentage of total macrophages, and together with anti‐mouse CD206 antibody gating by anti‐mouse CD68 antibody to measure the percentage of M2, and then were determined by FACS analysis. (A): Dot plots of CD68 + CD206 + M2. (B): Percentage of CD68 + total macrophages. (C): Percentage of CD68 + CD206 + M2. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Statistical analysis was done by one‐way analysis of variance followed by the least significant difference test, n = 6. Abbreviations: ERC, endometrial regenerative cell; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Staining, Inhibition

Journal: Stem Cells Translational Medicine

Article Title: Stromal Cell‐Derived Factor‐1 Mediates Cardiac Allograft Tolerance Induced by Human Endometrial Regenerative Cell‐Based Therapy

doi: 10.1002/sctm.17-0091

Figure Lengend Snippet: Stromal cell‐derived factor‐1 (SDF‐1) mediates ERC‐based therapy in increasing the percentage of both regulatory T cell (Tregs) and regulatory B cell (Bregs) in allograft recipients. Splenocytes were collected from B6 recipients in each group at postoperative day 8. For the Tregs, the percentage of CD25 + Foxp3 + cells in CD4 + gating population was determined by fluorescence‐activated cell sorting analysis. (A): Dot plots of CD4 + CD25 + Foxp3 + T cells. (B): Percentage of CD4 + CD25 + Foxp3 + T cells. For the Bregs, the percentage of CD83 + (C) , CD1d + CD5 + (D) , or IL‐10 + (E) cells in CD19 + gating population was measured. *ERCs indicated inhibition the function of SDF‐1 by AMD3100. Statistical analysis was done by one‐way analysis of variance followed by the least significant difference test, n = 6. Abbreviations: ERC, endometrial regenerative cell; RAPA, rapamycin.

Article Snippet: Rapamycin (RAPA) (Wyeth Pharmaceuticals, Soochow, China) was dissolved in 100% olive oil and was subcutaneously injected to the recipient B6 mice (2 mg/kg/day) for 13 days directly after cardiac transplantation.

Techniques: Derivative Assay, Fluorescence, FACS, Inhibition

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: 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: 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

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