Journal: Molecular Medicine Reports

Article Title: Triamcinolone acetonide modulates TGF-β2-induced angiogenic and tissue-remodeling effects in cultured human retinal pigment epithelial cells

doi: 10.3892/mmr.2021.12442

Figure Lengend Snippet: Effects of TA on constitutive and TGF-β2-stimulated signaling activities in cultured human RPE cells. (A) Human R-50 cells were treated with TA at 10 µM under serum-supplemented or -starved conditions for the indicated periods. Total proteins were subjected to western blot analysis of p-Smad2, p-ERK1/2 and p-p38 MAPK. (B) Densitometric analysis of (A). (C) R-50 RPE cells received 2 h of serum starvation, followed by treatment with 10 ng/ml TGF-β2 in the presence or absence of 10 µM TA. (D) Densitometric analysis of (C). Data are presented as the mean ± SD from three independent experiments. # P<0.05 vs. respective 0 h group; *P<0.05 vs. respective TA (−) group. RPE, retinal pigment epithelial; TA, triamcinolone acetonide; TGF-β2, transforming growth factor-β2; p, phosphorylated; ERK1/2, extracellular-regulated kinase 1/2; MAPK, mitogen-activated protein kinase.

Article Snippet: Rabbit monoclonal antibodies against total Smad2 (cat. no. 5339s), phosphorylated (p)-Smad2 (Ser465/467; cat. no. 3108s), total ERK1/2 (cat. no. 4695s), p-ERK1/2 (Thr202/185 and Tyr204/187; cat. no. 9101s), total p38 MAPK (cat. no. 8690s) and p-p38 MAPK (Thr180/Tyr182; cat. no. 4631) were purchased from Cell Signaling Technology, Inc.

Techniques: Cell Culture, Western Blot

Journal: Molecular Medicine Reports

Article Title: Triamcinolone acetonide modulates TGF-β2-induced angiogenic and tissue-remodeling effects in cultured human retinal pigment epithelial cells

doi: 10.3892/mmr.2021.12442

Figure Lengend Snippet: Suppressive effect of TA on TGF-β2-induced de novo synthesis of VEGF in cultured human retinal pigment epithelial cells. (A) Human R-50 cells were treated with TGF-β2 for 6 h in the presence of TA at the indicated doses or 0.1% MeOH as a SC. Total RNA was extracted and a multiplex reverse transcription-PCR was performed to simultaneously detect three isoforms of VEGF transcripts. (A) Representative gel showing VEGF isoform PCR products. (B) Densitometric analysis of the two major VEGF 121 and VEGF 165 transcripts. The density values of genes were normalized to those of the respective internal control, β-actin. Data are presented as the mean ± SD. (C) Reverse transcription-quantitative PCR analysis of the mRNA expression levels of VEGF 165 . TGF-β receptor blocker SB4 at 10 µM was used to block receptor kinase activity. (D) ELISA of R-50 cells receiving 10 ng/ml TGF-β2 for 48 h in the presence of 1 µM (TA1) or 10 µM (TA10) TA. (E) ELISA of R-50 cells receiving 10 ng/ml TGF-β2 for 24 h in the presence of p38 mitogen-activated protein kinase inhibitor SB2, MEK inhibitor PD, TGF-β receptor blocker SB4 at 10 µM or equivalent DMSO as a SC. Data are presented as the mean ± SD from three independent experiments. # P<0.05, ## P<0.01 vs. NC; *P<0.05, **P<0.01, ***P<0.001 vs. MeOH or vs. DMSO SC. TA, triamcinolone acetonide; TGF-β2, transforming growth factor-β2; VEGF, vascular endothelial growth factor; NC, negative control; SC, solvent control; SB4, SB431542; SB2, SB203580; PD, PD98059.

Article Snippet: Rabbit monoclonal antibodies against total Smad2 (cat. no. 5339s), phosphorylated (p)-Smad2 (Ser465/467; cat. no. 3108s), total ERK1/2 (cat. no. 4695s), p-ERK1/2 (Thr202/185 and Tyr204/187; cat. no. 9101s), total p38 MAPK (cat. no. 8690s) and p-p38 MAPK (Thr180/Tyr182; cat. no. 4631) were purchased from Cell Signaling Technology, Inc.

Techniques: Cell Culture, Multiplex Assay, Real-time Polymerase Chain Reaction, Expressing, Blocking Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Molecular Medicine Reports

Article Title: Triamcinolone acetonide modulates TGF-β2-induced angiogenic and tissue-remodeling effects in cultured human retinal pigment epithelial cells

doi: 10.3892/mmr.2021.12442

Figure Lengend Snippet: Effect of TA treatment on TGF-β2-driven ECM remodeling in cultured human retinal pigment epithelial cells. (A) Western blot analysis of signaling pathway proteins involved in TGF-β2-driven ECM remodeling. Human R-50 cells were treated with 10 ng/ml TGF-β2 for 24 h in the presence of either 10 µM p38 mitogen-activated protein kinase inhibitor SB2, MEK inhibitor PD or equivalent DMSO as a SC. Bands were densitometrically semi-quantified and the relative expression levels of (B) COL1A1, (C) α-SMA, (D) MMP-9 and (E) MMP-2 were normalized to internal actin expression levels. (F) R-50 cells were treated with 10 ng/ml TGF-β2 for 48 h in the presence of 10 µM TA, TGF-β receptor blocker SB4 or 0.1% methanol as a SC. The relative expression levels of (G) COL1A1, (H) α-SMA, (I) MMP-9 and (J) MMP-2 were densitometrically semi-quantified and normalized to β-actin. Fold changes are expressed as the mean ± SD from three independent experiments. # P<0.05, ## P<0.01 vs. NC; *P<0.05, **P<0.01 vs. SC. TA, triamcinolone acetonide; TGF-β2, transforming growth factor-β2; α-SMA, α-smooth muscle actin; MMP, matrix metalloproteinase; NC, negative control; SC, solvent control; SB4, SB431542; SB2, SB203580; PD, PD98059; COL1A1, collagen type I α1 chain.

Article Snippet: Rabbit monoclonal antibodies against total Smad2 (cat. no. 5339s), phosphorylated (p)-Smad2 (Ser465/467; cat. no. 3108s), total ERK1/2 (cat. no. 4695s), p-ERK1/2 (Thr202/185 and Tyr204/187; cat. no. 9101s), total p38 MAPK (cat. no. 8690s) and p-p38 MAPK (Thr180/Tyr182; cat. no. 4631) were purchased from Cell Signaling Technology, Inc.

Techniques: Cell Culture, Western Blot, Expressing, Negative Control

Journal: Molecular Medicine Reports

Article Title: Triamcinolone acetonide modulates TGF-β2-induced angiogenic and tissue-remodeling effects in cultured human retinal pigment epithelial cells

doi: 10.3892/mmr.2021.12442

Figure Lengend Snippet: Effect of TA treatment on TGF-β2-driven MMP-9 gelatinolytic activity in cultured human retinal pigment epithelial cells. (A) Human R-50 cells were treated with TGF-β2 for 48 h in the presence of 1 µM (TA1) or 10 µM (TA10) TA or 0.1% MeOH as a SC. The supernatants were collected and subjected to gelatin zymography detection of MMP-9 activity. Bands were densitometrically semi-quantified and the scanned densities were subtracted from the background levels in the M group. (B) R50 cells were treated with TGF-β2 for 24 h in the presence of either 10 µM p38 mitogen-activated protein kinase inhibitor SB, MEK inhibitor PD or equivalent DMSO as a SC. Fold changes presented as the mean ± SD were obtained from three independent experiments. # P<0.05 vs. NC; *P<0.05 vs. MeOH or SC. TA, triamcinolone acetonide; TGF-β2, transforming growth factor-β2; MMP, matrix metalloproteinase; NC, negative control; SC, solvent control; M, medium; MeOH, methanol; SB, SB203580; PD, PD98059.

Article Snippet: Rabbit monoclonal antibodies against total Smad2 (cat. no. 5339s), phosphorylated (p)-Smad2 (Ser465/467; cat. no. 3108s), total ERK1/2 (cat. no. 4695s), p-ERK1/2 (Thr202/185 and Tyr204/187; cat. no. 9101s), total p38 MAPK (cat. no. 8690s) and p-p38 MAPK (Thr180/Tyr182; cat. no. 4631) were purchased from Cell Signaling Technology, Inc.

Techniques: Activity Assay, Cell Culture, Zymography, Negative Control

Journal: Journal of Clinical Medicine

Article Title: Changes in Thyroid Hormone Signaling Mediate Cardiac Dysfunction in the Tg197 Mouse Model of Arthritis: Potential Therapeutic Implications

doi: 10.3390/jcm10235512

Figure Lengend Snippet: Reduced SERCA and increased activation of p38 MAPK in Tg197 LV samples. Densitometric assessment in arbitrary units and representative images of SERCA2a protein expression ( A ), PLB protein expression ( B ) and phosphorylated and total p38 MAPK ( C ) in left ventricle of WT C57BL/6JxCBA (WT, n = 4 for each gender) and Tg197 ( n = 6 for each gender) male and female mice. Values are represented in mean ± SEM; * p < 0.05. (SERCA: Sarcoplasmic Reticulum–Calcium ATPase, PLB: Phospholamban, LV: left ventricle).

Article Snippet: After Western blotting, filters were probed with specific antibodies against TRα1 (ab53729, Abcam, Cambridge, United Kingdom), TRβ1 (MA1-216, Affinity BioReagents, Colorado, USA), histone H3 (#9715, Cell Signaling Technology, MA, USA), SERCA (MA3-919, Affinity BioReagents), phospholamban (MA3-922, Affinity BioReagents), total and phosphorylated–ERK, (p-ERK) (#9101, #9102, Cell Signaling Technology), total and phosphorylated-Akt, (p-Akt) (#9271, #9272, Cell Signaling Technology), total and phosphorylated-p38 MAPK (#9211, #9212, Cell Signaling Technology), total and phosphorylated-JNKs (#9251, #9252, Cell Signaling Technology), and actin (A5316, Sigma Aldrich, MO, USA) all at concentration 1:1000 and overnight incubation at 4 °C.

Techniques: Activation Assay, Expressing

Journal: Journal of Clinical Medicine

Article Title: Changes in Thyroid Hormone Signaling Mediate Cardiac Dysfunction in the Tg197 Mouse Model of Arthritis: Potential Therapeutic Implications

doi: 10.3390/jcm10235512

Figure Lengend Snippet: Reduction of TRβ1 in Tg197 LV samples and female-specific reduction of Akt activation. Densitometric assessment in arbitrary units and representative Western blots of thyroid hormone receptor α1 nuclear expression (TRα1, A ), thyroid hormone receptor β1 nuclear expression (TRβ1, B ) and phosphorylated and total p38 MAPK ( C ) in left ventricle of WTC57BL/6JxCBA (WT, n = 4 per gender) and Tg197 ( n = 6 per gender) male and female mice. Values are represented in mean ± SEM; * p < 0.05 (H3: Histone 3).

Article Snippet: After Western blotting, filters were probed with specific antibodies against TRα1 (ab53729, Abcam, Cambridge, United Kingdom), TRβ1 (MA1-216, Affinity BioReagents, Colorado, USA), histone H3 (#9715, Cell Signaling Technology, MA, USA), SERCA (MA3-919, Affinity BioReagents), phospholamban (MA3-922, Affinity BioReagents), total and phosphorylated–ERK, (p-ERK) (#9101, #9102, Cell Signaling Technology), total and phosphorylated-Akt, (p-Akt) (#9271, #9272, Cell Signaling Technology), total and phosphorylated-p38 MAPK (#9211, #9212, Cell Signaling Technology), total and phosphorylated-JNKs (#9251, #9252, Cell Signaling Technology), and actin (A5316, Sigma Aldrich, MO, USA) all at concentration 1:1000 and overnight incubation at 4 °C.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Clinical Medicine

Article Title: Changes in Thyroid Hormone Signaling Mediate Cardiac Dysfunction in the Tg197 Mouse Model of Arthritis: Potential Therapeutic Implications

doi: 10.3390/jcm10235512

Figure Lengend Snippet: Reversal of molecular phenotype of cardiac dysfunction in Tg197 female mice upon treatment with T3. Densitometric assessment in arbitrary units and representative images of SERCA2a protein expression ( A ), PLB protein expression ( B ), phosphorylated and total Akt ( C ) and phosphorylated and total p38 MAPK ( D ) in left ventricle of TgFem + Placebo versus TgFem + Τ3 female mice. * p < 0.05. (SERCA: Sarcoplasmic Reticulum–Calcium ATPase, PLB: Phospholamban, LV: left ventricle).

Article Snippet: After Western blotting, filters were probed with specific antibodies against TRα1 (ab53729, Abcam, Cambridge, United Kingdom), TRβ1 (MA1-216, Affinity BioReagents, Colorado, USA), histone H3 (#9715, Cell Signaling Technology, MA, USA), SERCA (MA3-919, Affinity BioReagents), phospholamban (MA3-922, Affinity BioReagents), total and phosphorylated–ERK, (p-ERK) (#9101, #9102, Cell Signaling Technology), total and phosphorylated-Akt, (p-Akt) (#9271, #9272, Cell Signaling Technology), total and phosphorylated-p38 MAPK (#9211, #9212, Cell Signaling Technology), total and phosphorylated-JNKs (#9251, #9252, Cell Signaling Technology), and actin (A5316, Sigma Aldrich, MO, USA) all at concentration 1:1000 and overnight incubation at 4 °C.

Techniques: Expressing

Journal: The Journal of Neuroscience

Article Title: A Role for p38 Mitogen-Activated Protein Kinase in the Regulation of the Serotonin Transporter: Evidence for Distinct Cellular Mechanisms Involved in Transporter Surface Expression

doi: 10.1523/JNEUROSCI.3754-04.2005

Figure Lengend Snippet: Regulation of 5-HT uptake by p38 MAPK inhibitor PD169316. A, Dose dependence of PD169316 on 5-HT uptake. Synaptosomes (50 μg) were preincubated with various concentrations of PD169316 for 1 hr at 37°C followed by 3 min 5-HT uptake assays. Controls received the same volume of vehicle (DMSO). B, Time course effect of PD169316 on 5-HT uptake. Purified midbrain synaptosomes (50 μg) were preincubated with 10 μm PD169316 for the times indicated and then assayed for 5-HT (20 nm) uptake using a 3 min uptake time at 37°C (as described in Materials and Methods). Nonspecific uptake was defined as the uptake in the presence of 0.1 μm fluoxetine and subtracted from the total accumulation to yield specific uptake. The results were expressed as a percentage of uptake relative to the uptake observed in vehicle-treated synaptosomes, and data represent mean ± SEM of three experiments performed in triplicate. #p < 0.05, *p < 0.01 compared with vehicle control by one-way ANOVA with Bonferroni post hoc analysis. C, Specificity of p38 MAPK inhibition on SERT function. Synaptosomes were treated for 30 min at 37°C with the following MAPK inhibitors: PD169316 (20 μm), SB203580 (10 μm), PD98059 (50 μm), U0126 (50 μm), and SP600125 (50 μm). 5-HT uptake was measured as described in A. D, Constitutive expression of p38 MAPK in untreated synaptosomes and the effect of MAPK inhibitors on p38 MAPK activity. Synaptosomes were treated with various MAPK inhibitors as described above in C. After treatments, proteins were extracted from synaptosomes using RIPA buffer and subjected to SDS-PAGE (as described in Materials and Methods). Immunoblotting was performed using specific antibodies to p38 MAPK, MAPKAPK, phospho-p38 MAPK, and phospho-MAPKAPK as indicated by arrows. Representative immunoblots from three independent experiments are shown.

Article Snippet: Antibody to total p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA). siRNA SMART pool for p38 MAPK and scrambled siRNAs were purchased from Upstate (Charlottesville, VA).

Techniques: Purification, Control, Inhibition, Expressing, Activity Assay, SDS Page, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Role for p38 Mitogen-Activated Protein Kinase in the Regulation of the Serotonin Transporter: Evidence for Distinct Cellular Mechanisms Involved in Transporter Surface Expression

doi: 10.1523/JNEUROSCI.3754-04.2005

Figure Lengend Snippet: Induction of SERT activity in HEK-293 cells transfected with the active form of MKK3b(E). Cells were transfected with hSERT plus either pcDNA3 (empty vector) or the active form of MKK3b(E) (as described in Materials and Methods). After 24 hr, cells were washed once with KRH buffer and treated with PD169316 or the vehicle for 30 min at 37°C. A, Western blot analysis of phosphorylated form of p38 MAPK and MAPKAPK and total p38 MAPK and MAPKAPK. 5-HT uptake assays (B), SERT expression using biotinylation procedures (C), and quantitative SERT surface band density (D) were performed (as described in Materials and Methods and in the legends to Figs. ​Figs.11 and ​and4).4). The data shown in B and D are averages from three separate experiments, and data represent mean ± SEM performed in triplicate. Values significantly different from SERT plus pcDNA3-transfected cells (#p < 0.01; Student's t test); significant differences compared with vehicle-treated controls (*p < 0.05; Student's t test). Representative blots from three independent experiments are shown in A and C.

Article Snippet: Antibody to total p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA). siRNA SMART pool for p38 MAPK and scrambled siRNAs were purchased from Upstate (Charlottesville, VA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: The Journal of Neuroscience

Article Title: A Role for p38 Mitogen-Activated Protein Kinase in the Regulation of the Serotonin Transporter: Evidence for Distinct Cellular Mechanisms Involved in Transporter Surface Expression

doi: 10.1523/JNEUROSCI.3754-04.2005

Figure Lengend Snippet: Effect of reducing p38 MAPK expression by siRNAs on 5-HT transport. HEK-293 cells were transiently transfected with 200 nm siRNAs targeted to p38 MAPK or scrambled siRNAs before the transfection with hSERT (as described in Materials and Methods). After 24 hr, cells were used for 5-HT transport, p38 MAPK expression, and SERT biotinylation as in Figure 9. A, A representative Western blot from three separate experiments shows the expression levels of total and phospho-p38 MAPK and calnexin. B, 5-HT uptake values are averages from three separate experiments, and data represent mean ± SEM. C, D, SERT expression measurements using biotinylation procedures and quantitation of SERT surface band densities were performed (as described in Materials and Methods and in the legends to Figs. ​Figs.11 and ​and4).4). Representative blots from three independent experiments are shown in C. The data shown in D are averages of band densities from three separate experiments, and data represent mean ± SEM. Values significantly different from SERT plus scrambled siRNAs transfected cells (*p < 0.05; Student's t test).

Article Snippet: Antibody to total p38 MAPK was from Santa Cruz Biotechnology (Santa Cruz, CA). siRNA SMART pool for p38 MAPK and scrambled siRNAs were purchased from Upstate (Charlottesville, VA).

Techniques: Expressing, Transfection, Western Blot, Quantitation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Si-Miao-Yong-An decoction attenuates cardiac fibrosis via suppressing TGF-β1 pathway and interfering with MMP-TIMPs expression.

doi: 10.1016/j.biopha.2020.110132

Figure Lengend Snippet: Fig. 5. SMYAD improved cardiac fibrosis by inhibiting TGF-β1/TAK1/P38 pathway and blocked pressure overload-induced TGF-β mRNA expression. (A) tgfb1, (B) tgfb2, (C) tgfb3, (D) tgfbr1 mRNA levels in different group. (E) The p-TAK1 and p-p38 protein level in sham, TAC, TAC + Captopril and TAC + SMYAD group. Western blot analysis of (F) p-TAK1/TAK1 and (G) p-p38/p38. Sham (n=6), TAC (n=6), TAC + Captopril (n=6), TAC + SMYAD (n=6). ### P < 0.001, ## P < 0.01, # P < 0.05 vs the Sham group; *** P < 0.001, ** P < 0.01, * P < 0.05 vs the TAC group.

Article Snippet: After blocking with 5 % non-fat milk for 1 h at room temperature, the blots were incubated overnight at 4 °C with the following antibodies: Col1 (Collagen Type I, 1:1000, Proteintech Cat 14695-1-AP); Col3 (Collagen Type III (N-Terminal), 1:1000, Proteintech Cat 22734-1-AP); αSMA (α-Smooth Muscle Actin, 1:1000, Cell Signaling Technology Cat 19245); MMP1 (MMP1, 1:1000, Proteintech Cat 10371-2-AP); MMP9 (MMP9 (N-Terminal), 1:1000, Proteintech Cat 10375-2-AP); TIMP1 (TIMP1, 1:1000, ab179580, Abcam); TIMP2 (TIMP2, 1:1000, ab180630, Abcam); CTGF (CTGF, 1:1000, Proteintech Cat 23936-1-AP); TGFβ1, (TGFβ1, 1:1000, Cell Signaling Technology Cat 3711); p-SMAD2 (Phospho-SMAD2, 1:500, Cell Signaling Technology Cat 18338); t-SMAD2 (SMAD2, 1:1000, Proteintech Cat 12570-1-AP); p-SMAD3 (Phospho-SMAD3, 1:500, Cell Signaling Technology Cat 9520); t-SMAD3 (SMAD3, 1:1000, Proteintech Cat 25494-1-AP); SMAD4 (SMAD4, 1:1000, Proteintech Cat 10231-1-AP); SMAD7 (SMAD7, 1:1000, Proteintech Cat 25840-1-AP); p-TAK1 (Phospho-TAK1, 1:1000, Cell Signaling Technology Cat 9339); t-TAK1 (TAK1, 1:1000, Cell Signaling Technology Cat 5206); p-p38 (Phospho-p38 MAPK, 1:1000, Cell Signaling Technology Cat 4511); t-p38 (P38 MAPK, 1:1000, Proteintech Cat 14064-1-AP); GAPDH (1:1,000, Cell Signaling Technology Cat 2118) was used as protein loading controls for shown in the figures.

Techniques: Expressing, Western Blot