Journal: Molecules

Article Title: The Preventive Effect of Specific Collagen Peptides against Dexamethasone-Induced Muscle Atrophy in Mice

doi: 10.3390/molecules28041950

Figure Lengend Snippet: Effect of CP on myokine alterations. ( A – D ) The mRNA expression of myostatin ( A ), TGF-β ( B ), BDNF ( C ), and FNDC5 ( D ) in the muscles was assessed using real-time PCR. ( E , F ) Total Smad2/3, phospho-Smad2, total-AKT, phospho-AKT, and GAPDH levels in the muscles were assessed by immunoblotting. Total proteins were stained by Ponceau S. ImageJ was used to measure and quantify the band intensity. Arrow in ( E ) indicates p-Smad 2, and this band was used to quantify the p-Smad2 level. The p-Smad2 and p-AKT levels were normalized by Ponceau S staining. ( G ) Total and phospho-mTOR levels were analyzed by ELISA. The p-mTOR levels were normalized against the total mTOR levels. Data are presented as the means ± SD of values obtained from seven biological replicates. # p < 0.05, ## p < 0.01, DEX vs. control. # p < 0.05, ## p < 0.01, DEX + CP (0.25 and 0.5) vs. control. * p < 0.05, ** p < 0.01, DEX + CP (0.25 and 0.5) vs. DEX; CP, collagen peptides; DEX, dexamethasone.

Article Snippet: To quantify the activity of mTOR and p-mTOR, Pathscan sandwich ELISA kits (mTOR: CST#7974, p-mTOR: CST#7976) were used.

Techniques: Expressing, Muscles, Real-time Polymerase Chain Reaction, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Molecules

Article Title: The Preventive Effect of Specific Collagen Peptides against Dexamethasone-Induced Muscle Atrophy in Mice

doi: 10.3390/molecules28041950

Figure Lengend Snippet: Anti-inflammatory activity of CP in muscle tissue. ( A ) Evans blue staining of the calf muscle. The blue region in the representative images indicates damaged muscle fibers. The Evans blue-positive area was quantified and is presented as a relative percentage of the control below the representative photos. This experiment was performed with three mice per group. ( B , C ) The mRNA expression of TNF-α and IL-1β in the muscles was assessed using real-time PCR. Data are presented as the means ± SD of values obtained from seven biological replicates. ( D ) The IL-1β levels in serum were examined using ELISA. Data are presented as the means ± SD of values obtained from three biological replicates. # p < 0.05, ## p < 0.01, DEX vs. control. # p < 0.05, ## p < 0.01, DEX + CP (0.25 and 0.5) vs. control. * p < 0.05, ** p < 0.01, DEX + CP (0.25 and 0.5) vs. DEX; CP, collagen peptides; DEX, dexamethasone.

Article Snippet: To quantify the activity of mTOR and p-mTOR, Pathscan sandwich ELISA kits (mTOR: CST#7974, p-mTOR: CST#7976) were used.

Techniques: Activity Assay, Staining, Control, Expressing, Muscles, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: Antifatigue Effects of Antrodia cinnamomea Cultured Mycelium via Modulation of Oxidative Stress Signaling in a Mouse Model

doi: 10.1155/2017/9374026

Figure Lengend Snippet: Three-week AC treatment upregulated the levels of phosphor-Akt and phosphor-AMPK and suppressed the expression of phosphor-mTOR in (a) livers and (b) skeletal muscle of mice with 60 min swimming. The data on quantified protein expression were normalized to the expressions of GAPDH. The data are expressed as means ± SEM ( n = 6) and analyzed using a one-way ANOVA ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 in a comparison with the control mice.

Article Snippet: Immunoblotting was detected using primary antibodies including phosphor- (P-) Akt (07-1398), total- (T-) mTOR (04-385), P-mTOR (09-213) (Merck Millipore, Darmstadt, Germany), T-Akt (ab131443), T-AMPK (ab133348), and P-AMPK (ab133348) (Abcam, Cambridge, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-25778) (Santa Cruz Biotechnology, Santa Cruz, USA) via incubation overnight at 4°C followed by washing in TBST buffer containing 5% BSA and 0.1% Tween-20.

Techniques: Expressing, Comparison, Control

Journal: Molecular Medicine Reports

Article Title: Curcumin induces apoptosis by inhibiting BCAT1 expression and mTOR signaling in cytarabine-resistant myeloid leukemia cells

doi: 10.3892/mmr.2021.12204

Figure Lengend Snippet: Curcumin inhibits BCAT1 expression and mTOR signaling in R-HL60 cells. (A) R-HL60 cells were treated with 0–50 µM curcumin for 24 h. (B) R-HL60 cells were treated with curcumin at a concentration of 50 µM for 2–48 h. Curcumin caused the reduction of the ratio of p-mTOR/t-mTOR and BCAT1 protein expression levels. The data represent the mean ± SEM of three independent experiments with triple replicates and were analyzed using the one-way ANOVA test to determine the differences of multiple groups. Bonferroni was used as a post hoc test. *P<0.05 the experimental group vs. the vehicle control group. BCAT1, branched-chain amino acid transaminase 1; R-, resistant; p-, phosphorylated; t-, total.

Article Snippet: The membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight with primary antibodies against human phosphorylated (p)-mTOR (ser2448; 1:1,000; Arigo Biolaboratories; cat. no. ARG40666), total (t)-mTOR (1:1,000; Arigo Biolaboratories; cat. no. ARG57640), BCAT1 (1:3,000; Cell Signaling Technology, Inc.; cat. no. 12822), poly (ADP-ribose) polymerase 1 (PARP 1; 1:2,000; Abcam; cat. no. ab32138), cleaved (c)-PARP 1 (1:15,000; Abcam; cat. no. ab32064) and GAPDH (1:30,000; Ambion; Thermo Fisher Scientific, Inc.; cat. no. AM4300) at 4°C.

Techniques: Expressing, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: Curcumin induces apoptosis by inhibiting BCAT1 expression and mTOR signaling in cytarabine-resistant myeloid leukemia cells

doi: 10.3892/mmr.2021.12204

Figure Lengend Snippet: mTOR and BCAT1 pathways can modulate each other and regulate apoptosis. R-HL60 cells were treated with (A) 10 µM PP242 for 24 h or (B) 80 µM BCATc inhibitor 2 for 48 h, and p-mTOR, t-mTOR, PARP1, c-PARP1 and BCAT1 protein expression levels were detected using western blotting. Both PP242 and BCATc inhibitor 2 significantly decreased the ratio of p-mTOR/t-mTOR, PARP1 and BCAT1 expression and significantly induced c-PARP1 expression. The data were compared with the vehicle control group (DMSO) and represented the mean ± SEM of three independent experiments with triple replicates per experiment. Paired t-tests was used to determine the differences between the experimental and control groups. *P<0.05 the experimental group vs. the vehicle control group. BCATi, BCATc inhibitor 2; BCAT, branched-chain amino acid transaminase; R-, resistant; p-, phosphorylated; t-, total; c-, cleaved; PARP 1, poly (ADP-ribose) polymerase 1.

Article Snippet: The membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight with primary antibodies against human phosphorylated (p)-mTOR (ser2448; 1:1,000; Arigo Biolaboratories; cat. no. ARG40666), total (t)-mTOR (1:1,000; Arigo Biolaboratories; cat. no. ARG57640), BCAT1 (1:3,000; Cell Signaling Technology, Inc.; cat. no. 12822), poly (ADP-ribose) polymerase 1 (PARP 1; 1:2,000; Abcam; cat. no. ab32138), cleaved (c)-PARP 1 (1:15,000; Abcam; cat. no. ab32064) and GAPDH (1:30,000; Ambion; Thermo Fisher Scientific, Inc.; cat. no. AM4300) at 4°C.

Techniques: Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: Curcumin induces apoptosis by inhibiting BCAT1 expression and mTOR signaling in cytarabine-resistant myeloid leukemia cells

doi: 10.3892/mmr.2021.12204

Figure Lengend Snippet: Schematic of curcumin regulating apoptosis via the BCAT1 and mTOR pathways. BCAT1, branched-chain amino acid transaminase 1.

Article Snippet: The membranes were blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight with primary antibodies against human phosphorylated (p)-mTOR (ser2448; 1:1,000; Arigo Biolaboratories; cat. no. ARG40666), total (t)-mTOR (1:1,000; Arigo Biolaboratories; cat. no. ARG57640), BCAT1 (1:3,000; Cell Signaling Technology, Inc.; cat. no. 12822), poly (ADP-ribose) polymerase 1 (PARP 1; 1:2,000; Abcam; cat. no. ab32138), cleaved (c)-PARP 1 (1:15,000; Abcam; cat. no. ab32064) and GAPDH (1:30,000; Ambion; Thermo Fisher Scientific, Inc.; cat. no. AM4300) at 4°C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Sirolimus Suppresses Phosphorylation of Cofilin and Reduces Interstitial Septal Thickness in Sporadic Lymphangioleiomyomatosis

doi: 10.3390/ijms22168564

Figure Lengend Snippet: Effect of sirolimus on decreasing mTOR expression. Representative quantifications of the immunoblot densitometries of protein expression are shown for lungs from the two groups. mTOR (289-kDa band) expression was increased in the lung tissues of LAM patients, and this change was reversed by administering sirolimus (LAM/sirolimus). HMB45 (55-kDa band) expression was increased in the lung tissues of LAM patients but did not decrease significantly after sirolimus administration (LAM/sirolimus), relative (Rel.). The bars represent the mean ±SEM for n = 3-4 samples. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the non-LAM group. # p < 0.05 compared with the LAM group without sirolimus treatment.

Article Snippet: For Western blotting, immunoblotting was performed using anti-HMB45 (Santa Cruz Biotechnology, SC-59305, Dallas, TX, USA), anti-total mTOR (EnoGene, Ab-2481, New York, NY, USA), and anti-p-cofilin (Cell Signaling, 8354, Danvers, MA, USA) as primary antibodies.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Sirolimus Suppresses Phosphorylation of Cofilin and Reduces Interstitial Septal Thickness in Sporadic Lymphangioleiomyomatosis

doi: 10.3390/ijms22168564

Figure Lengend Snippet: Effect of sirolimus on LAM cell proliferation and migration associated with mTOR and p-cofilin expression. ( a ) The LAM cell clusters (size between 70 μm to 100 μm) was isolated from LAM patients, the phase contrast images show the morphology of LAM cells in adherent culture at day 1, day 2, and day 3. The images were obtained using 40× objectives. ( b ) The primary culture LAM cells were identified by the specific markers HMB45 to characterize the LAM cells. Immunocytochemistry showing HMB45 localization (brown staining; nuclear staining with hematoxylin in blue). ( c ) LAM cells were treated with or without sirolimus (0.1 μM). Western blot was used to assess the protein expression levels in whole-cell extracts. ( d ) Relative expression values of mTOR, pCofilin, tCofilin, desmin, and SMA obtained by densitometry. The data are presented as the mean ±SEM of three samples in each group. ** p < 0.01 and *** p < 0.001 compared with LAM cells without sirolimus. ( e ) cell viability, ( f ) Sirolimus treatment inhibited the proliferation of LAM cells. ( g ) Sirolimus treatment inhibited the migration of LAM cells. The data (mean ±SEM [ n = 4]) are presented as the fold change in cell numbers compared with those in the untreated group. *** p < 0.001 compared with the untreated group. ( h ) the schema: possible mechanisms of mTOR inhibitor in LAM via decreased p-cofilin.

Article Snippet: For Western blotting, immunoblotting was performed using anti-HMB45 (Santa Cruz Biotechnology, SC-59305, Dallas, TX, USA), anti-total mTOR (EnoGene, Ab-2481, New York, NY, USA), and anti-p-cofilin (Cell Signaling, 8354, Danvers, MA, USA) as primary antibodies.

Techniques: Migration, Expressing, Isolation, Immunocytochemistry, Staining, Western Blot