Journal: Scientific Reports

Article Title: Anti-fibrogenic effect of umbilical cord–derived mesenchymal stem cell–conditioned media in human esophageal fibroblasts

doi: 10.1038/s41598-024-73091-7

Figure Lengend Snippet: UC-MSC-CM decreases the TGF-β1-induced phosphorylation of Smad2. HEFs were treated with TGF-β1 (5 ng/mL) and cultured with or without UC-MSC-CM for 48 h. ( A ) Representative western blots showing the protein expression of phosphorylated Smad2 (p-Smad2) and total Smad2 with GAPDH as a loading control. ( B ) Quantitation of p-Smad2 using western blot analyses (n = 4). Data are expressed as the mean ± SEM. # P < 0.05 versus the control; *P < 0.05 versus TGF-β1 treatment only (ANOVA w/ Tukey). Original images of blots are presented in Supplementary Fig. S5.

Article Snippet: Primary antibodies against procollagen1A1 (Procol1A1) (SP1D8, Developmental Studies Hybridoma Bank, Iowa City, IA), fibronectin (FN) (ab2413, Abcam, Cambridge, MA), α-SMA (A2547, Sigma), phospho-Smad2 (Ser465/467) (#3108, Cell Signaling), total-Smad2 (#3102, Cell Signaling), RhoA (#sc-418, Santa Cruz Biotechnology, Dallas, TX), GAPDH (#2118, Cell Signaling), Mkl1(MRTF-A) (21166-1-AP, ProteinTech), SRF (#5147, Cell Signaling), HDAC1 (#5356, Cell Signaling), phospho-YAP (Ser127) (#13008, Cell Signaling), YAP (#sc-101199, Santa Cruz Biotechnology, Dallas, TX), and TAZ (#8418, Cell Signaling) were used.

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Expressing, Control, Quantitation Assay

Journal: Scientific Reports

Article Title: Anti-fibrogenic effect of umbilical cord–derived mesenchymal stem cell–conditioned media in human esophageal fibroblasts

doi: 10.1038/s41598-024-73091-7

Figure Lengend Snippet: Inhibition of TGF-β1-induced fibrogenic activation by UC-MSC-CM in HEFs. This model illustrates the cascade initiated by TGF-β1 binding to its receptor, which promotes RhoA activation that subsequently activates ROCK. Activation of the RhoA/ROCK pathway enhances G-actin polymerization into F-actin, releasing MRTF-A from G-actin, which then translocates into the nucleus. Concurrently, RhoA-driven F-actin polymerization leads to YAP/TAZ dephosphorylation and their nuclear translocation. In the nucleus, MRTF-A, YAP, and TAZ serve as transcriptional co-activators with SRF or TEAD to upregulate fibrogenic genes such as Procol1A1, FN, and α-SMA. Additionally, TGF-β1 induces phosphorylation of Smad2/3, which then bind to Smad4 and translocate into the nucleus, where they induce fibrosis. UC-MSC-CM is depicted as interfering with TGF-β1 signal transduction by impeding the Smad 2 pathway and the RhoA-mediated MRTF-A/SRF and YAP/TAZ pathways, thereby attenuating fibrogenic gene transcription.

Article Snippet: Primary antibodies against procollagen1A1 (Procol1A1) (SP1D8, Developmental Studies Hybridoma Bank, Iowa City, IA), fibronectin (FN) (ab2413, Abcam, Cambridge, MA), α-SMA (A2547, Sigma), phospho-Smad2 (Ser465/467) (#3108, Cell Signaling), total-Smad2 (#3102, Cell Signaling), RhoA (#sc-418, Santa Cruz Biotechnology, Dallas, TX), GAPDH (#2118, Cell Signaling), Mkl1(MRTF-A) (21166-1-AP, ProteinTech), SRF (#5147, Cell Signaling), HDAC1 (#5356, Cell Signaling), phospho-YAP (Ser127) (#13008, Cell Signaling), YAP (#sc-101199, Santa Cruz Biotechnology, Dallas, TX), and TAZ (#8418, Cell Signaling) were used.

Techniques: Inhibition, Activation Assay, Binding Assay, De-Phosphorylation Assay, Translocation Assay, Phospho-proteomics, Transduction

Journal: PLoS ONE

Article Title: Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation

doi: 10.1371/journal.pone.0042453

Figure Lengend Snippet: Fold change in glycogenin normalized by total protein content of 1 hour (▪) or 24 hours (▪) stimulated samples compared to control situation (□) measured by Western-blot of Glycogenin-1. The inset shows a representative blot for each situation (from 0 to 24 hours of rest): in the upper panel samples with 1 hour of CLFS, and the lower panel the groups with 24 hours of CLFS. C is the contralateral non-stimulated control sample of each sample showed. In the bar chart the control group was composed with the pool of all contralateral1 non-stimulated muscle samples results (n = 50). n = 5 for the stimulated and rested groups. Data are means ± SE. There was no statistical difference under any condition.

Article Snippet: The primary antibodies used were: muscle GS (mouse monoclonal from Chemicon, dilution 1∶2000), glycogenin (guinea pig polyclonal, dilution 1∶2000), AMPK-P (T172) (rabbit polyclonal from Cell Signalling, dilution 1∶1000), and AMPKα1 or AMPKα2 (goat polyclonal, dilution 1∶2000).

Techniques: Control, Western Blot

Journal: Cellular microbiology

Article Title: Plasmodium falciparum soluble extracts potentiate the suppressive function of polyclonal T regulatory cells through activation of TGFβ-mediated signals.

doi: 10.1111/j.1462-5822.2011.01622.x

Figure Lengend Snippet: Fig. 6. Effect of antibodies to P. falciparum TRAP and MMP inhibitor on TGFb signalling of CD25+ cells. Activated CD25+ cells were incubated in serum-free medium for 2 h in the presence of 10 mg ml-1 PfSEs (A) or uRBC lysates (B), pre-treated with mouse monoclonal anti-PfTRAP antibodies (1 mg ml-1) or anti-human TSP antibodies (1 mg ml-1) or control mouse IgG (1 mg ml-1) or GM 1489 (0.2 mg ml-1) or the inhibitor vehicle DMSO. Western blot analysis was performed by using anti-phospho-SMAD2/3 and anti-total SMAD 2/3 antibodies. The histograms represent the mean increase of SMAD 2/3 phosphorylation induced by PfSEs or uRBCs stimulation relative to unstimulated cultures in the presence of different inhibitors. Data from each experimental point were recorded as P-SMAD/T-SMAD. Statistical analysis was performed by Student’s t-test.

Article Snippet: Membranes were stripped at 56°C and stained with 1:1000 a-total SMAD2 (Cell Signaling, Cambridge, UK).

Techniques: Incubation, Control, Western Blot, Phospho-proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of IL-1β on the BMP/Smad signaling pathway in mouse bone marrow mesenchymal stem cells. (A) Levels of p-Smad1/5, overall Smad1, Smad5 and Smad4 at 7 days were examined via Western blot analysis. (B) Quantitative assay of p-Smad1/5/GAPDH. * P<0.05, ** P<0.01, *** P<0.001. p-, phosphorylated; IL, interleukin; BMP, bone morphogenetic protein.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: IL-1β promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells via the BMP/Smad pathway within a certain concentration range

doi: 10.3892/etm.2020.9065

Figure Lengend Snippet: Effect of TGF-β/Smad inhibitor on the osteogenic differentiation of MBMMSCs. MBMMSCs were treated with osteogenic differentiation medium in the presence of 0.1 ng/ml IL-1β along with the inhibitor of TGF-β/Smad LDN193189. (A) Western blot analysis results for the levels of p-Smad1/5, and total Smad1, Smad5 and Smad4 at 7 days. (B) Quantitative results of p-Smad1/5/GAPDH. (C) Entire plate views of ALP staining at 7 days and alizarin red staining at 21 days. (D) Quantitative evaluation of ALP activity and alizarin red staining results. (E) mRNAs expression levels of RUNX2, ALP, COL1A1, BSP, BMP2, OCN and OPN at 7 days. Expression levels were normalized to GAPDH. *** P<0.001. p-, phosphorylated; IL, interleukin; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; OCN, osteocalcin; OPN, osteopontin; BSP, bone sialoprotein; RUNX2, runt-related transcription factor 2; COL1A1, type I collagen; MBMMSCs, mouse bone marrow mesenchymal stem cells.

Article Snippet: After incubation in 5% skimmed milk for 1 h at 25˚C to block non-specific binding, the membranes were incubated with primary antibodies (1:1,000 dilution) against Smad1, Smad5, Smad4 and phosphorylated (p)-Smad1/5 (cat. no. 12656; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; Cell Signaling Technology, Inc.) for 8 h at 4˚C.

Techniques: Western Blot, Staining, Activity Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Sodium Tanshinone IIA Sulfonate Prevents Angiotensin II-Induced Differentiation of Human Atrial Fibroblasts into Myofibroblasts

doi: 10.1155/2018/6712585

Figure Lengend Snippet: DS-201 prevents Ang II-induced TGF- β 1 activation in human atrial fibroblasts. Atrial fibroblasts were exposed to Ang II (0.5 μ M) with or without DS-201 (0, 5, 25, 50, and 100 μ M) for 24 h. (a) Expression of TGF- β 1 was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ratio of TGF- β 1 normalized to GAPDH was calculated. (b) Phosphorylation (p) of Smad2/3 was analyzed by western blotting. Representative images of 3 independent experiments and densitometric analysis of phosphorylated Smad2/3 normalized to total Smad2/3 are shown. (c) Expression of periostin was analyzed by western blotting, and representative images of 3 independent experiments are shown. The ration of periostin normalized to GAPDH was calculated. ∗∗ P < 0.01 versus control (first bar); ## P < 0.01 versus Ang II.

Article Snippet: Antibodies to α -SMA, phosphorylated Smad2/3, and total Smad2/3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Expressing, Western Blot