Journal: International Journal of Medical Sciences

Article Title: Blockade of SK4 channels suppresses atrial fibrillation by attenuating atrial fibrosis in canines with prolonged atrial pacing

doi: 10.7150/ijms.69626

Figure Lengend Snippet: Effects of SK4 on the expression of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in atria . (A) Representative Western blot images of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 expression levels in the atria. (B-G) The mean levels of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in the atria. *** P<0.001 versus the sham group. ### P<0.001 versus the pacing group. ▲▲ P<0.01 versus the sham group. ▲▲▲ P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

Article Snippet: The membranes containing the sample proteins were incubated overnight with the primary antibodies against SK4 (Bioss, USA), collagen I, collagen III, matrix metalloproteinase (MMP)-2, MMP-9 (Abcam, USA), transforming growth factor β1 (TGF-β1) (Proteintech Group Inc., China), connective tissue growth factor (CTGF) (Proteintech Group Inc., China), Smad2 and phospho-specific Smad2 (p-Smad2), Smad3 and phospho-specific Smad3 (p-Smad3) , p38 and phospho-specific p38 (p-p38), ERK1/2 and phospho-specific ERK1/2 (p-ERK1/2) (Cell Signaling Technology, USA), c-Fos (Abcam, USA) and c-Jun (Abcam, USA) at 4 °C.

Techniques: Expressing, Western Blot

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: (A) Experimental strategy to delete Alk5 in ECs. Red arrows indicate the qPCR primer hybridization sites. (B) qPCR measurement of Alk5 levels in Alk5l/l and Alk5iEKO mouse brain ECs (mean ± SEM, each dot represents one mouse, **p < 0.01, Mann-Whitney U test). (C) Western blot for phospho-SMAD2/3, and β-actin using CTRL ( Alk5l/l ) and Alk5iEKO mouse brain ECs treated for 30 min with TGF-β1. (D) Top, experimental strategy to assess retinal vascularization at P6. Red arrows indicate TAM injections. Bottom, IsoB4 staining of retinal flat mounts. (E) Quantification of vascular parameters of Alk5l/l and Alk5iEKO P6 retinas (mean ± SEM, each dot represents one retina, *p < 0.05, Mann-Whitney U test). (F) Top, experimental strategy to assess retinal vascularization at P12. Red arrows indicate TAM injections. Bottom, IsoB4 staining of retinal flat mounts. (G) Quantification of vascular parameters of Alk5l/l and Alk5iEKO P12 retinas (mean ± SEM, each dot represents one retina, **p < 0.01, Mann-Whitney U test). (H) IsoB4 and DAPI double staining of P12 Alk5l/l and Alk5iEKO retina cross-sections. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptors.

Article Snippet: Proteins were detected using the following antibodies: p-SMAD2/3 (8828, Cell Signaling 1:1000), SMAD2 (5339, Cell Signaling 1:1000), SMAD3 (9523 Cell Signaling 1:1000) and β-actin (A1978, Sigma).

Techniques: Hybridization, MANN-WHITNEY, Western Blot, Staining, Double Staining

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: (A) Experimental strategy to delete Smad2 and Smad3 in ECs. Red arrows indicate TAM injections. (B and C) Western blot for SMAD2 or SMAD3 and β-actin using ECs isolated from lungs of WT ( Smad2l/l or Smad3l/l ) and Smad2iEKO and Smad3iEKO mice, respectively. (D) IsoB4/α-SMA double staining of P12 retinas of the indicated genotypes. (E) Quantification of vascular parameters of Smad2/3l/l and Smad2/3iEKO P12 retinas (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, Mann-Whitney U test). (F) Experimental strategy to delete Smad4 in ECs. Red arrows indicate TAM injections. (G) IsoB4 and α-SMA double staining of P12 Smad4l/l and Smad4iEKO retinas. (H) Quantification of vascular parameters of P12 retinas of the indicated genotypes (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, **p < 0.001, Mann-Whitney U test). (I) Double immunostainings for IsoB4 and APOE, VWF, TXNDR1, or PLVAP of retina flat mounts of the indicated genotypes at P12. A, artery; V, vein.

Article Snippet: Proteins were detected using the following antibodies: p-SMAD2/3 (8828, Cell Signaling 1:1000), SMAD2 (5339, Cell Signaling 1:1000), SMAD3 (9523 Cell Signaling 1:1000) and β-actin (A1978, Sigma).

Techniques: Western Blot, Isolation, Double Staining, MANN-WHITNEY

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Proteins were detected using the following antibodies: p-SMAD2/3 (8828, Cell Signaling 1:1000), SMAD2 (5339, Cell Signaling 1:1000), SMAD3 (9523 Cell Signaling 1:1000) and β-actin (A1978, Sigma).

Techniques: Recombinant, Fluorescence, Plasmid Preparation, Modification, Western Blot, SYBR Green Assay, Imaging, Multiplex Assay, Expressing, Software, Laser-Scanning Microscopy

Journal: PLoS ONE

Article Title: TGF-β1/Smad Signaling Pathway Regulates Epithelial-to-Mesenchymal Transition in Esophageal Squamous Cell Carcinoma: In Vitro and Clinical Analyses of Cell Lines and Nomadic Kazakh Patients from Northwest Xinjiang, China

doi: 10.1371/journal.pone.0112300

Figure Lengend Snippet: A . Effects of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL) on the expression of E-cadherin (molecular weight 97 kDa), N-cadherin (molecular weight 100 kDa), vimentin (molecular weight 57 kDa), p-Smad2 (molecular weight 52 kDa) and Smad7 (molecular weight 51 kDa) by Western Blot. B . Quantiative analysis of treatment of Eca109 cells with TGF-β1 (1, 5, or 10 ng/mL), E-cadherin, N-cadherin, vimentin, P-Smad2 and Smad7 expression levels; Y axis: banding densities of test marker versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01. C . Effects of SB431542 (1, 5, or 10 µM) on the expression of E-cadherin, N-cadherin, vimentin, Smad2/3 and Smad7 in Eca109 cells by Western Blot. D . Quantiative analysis of treatment of Eca109 cells with SB431542 (1, 5, or 10 µM), E-cadherin, vimentin, Smad2/3 and Smad7 expression levels; Y axis: banding densties of test versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01; Each experiment was repeated three times. E . Effects of treatment of Eca 9706 cells with TGF-β1 (1, 5, or 10 ng/mL) on the expression of E-cadherin, N-cadherin, vimentin, p-Smad2 by Western Blot. F . Quantiative analysis of treatment of Eca 9706 cells with TGF-β1 (1, 5, or 10 ng/mL), E-cadherin, N-cadherin, vimentin, P-Smad2 expression levels; Y axis: banding densities of test marker versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01. G . Effects of SB431542 (1, 5, or 10 µM) on the expression of E-cadherin, N-cadherin, vimentin, Smad2/3 in Eca 9706 cells by Western Blot. H . Quantiative analysis of treatment of Eca 9706 cells with SB431542 (1, 5, or 10 µM), E-cadherin, vimentin, Smad2/3 expression levels; Y axis: banding densties of test versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01; Each experiment was repeated three times. I . Effects of treatment of KYSE150 cells with TGF-β1 (1, 5, or 10 ng/mL) on the expression of E-cadherin, N-cadherin, vimentin, p-Smad2 by Western Blot. J . Quantiative analysis of treatment of KYSE150 cells with TGF-β1 (1, 5, or 10 ng/mL), E-cadherin, N-cadherin, vimentin, P-Smad2 expression levels; Y axis: banding densities of test marker versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01. K . Effects of SB431542 (1, 5, or 10 µM) on the expression of E-cadherin, N-cadherin, vimentin, Smad2/3 in KYSE150 cells by Western Blot. L . Quantiative analysis of treatment of KYSE150 cells with SB431542 (1, 5, or 10 µM), E-cadherin, vimentin, Smad2/3 expression levels; Y axis: banding densties of test versus β-actin. Data are expressed as a significant change relative to the control. Each bar represents the mean±s.d. *, p<0.05, **, p<0.01. Each experiment was repeated three times.

Article Snippet: The blots were incubated with primary antibodies overnight at 4°C with rabbit anti-p-Smad2 (1∶1000, Cell Signaling Technology, Massachusetts, USA), anti-Smad7 (1∶200, Santa Cruz Biotechnology, California, USA), anti-E-cadherin (1∶500, Abcam, Cambridge, United Kingdom), mouse anti-vimentin (1∶100, Santa Cruz Biotechnology, California USA), or anti-N-cadherin antibodies (1∶1000, Abcam, Cambridge, United Kingdom)This procedure was followed by incubation with sheep anti-mouse (1∶20000) or anti-rabbit (1∶20000) HRP-labeled secondary antibodies for 2 h at room temperature.

Techniques: Expressing, Molecular Weight, Western Blot, Marker

Journal: PLoS ONE

Article Title: TGF-β1/Smad Signaling Pathway Regulates Epithelial-to-Mesenchymal Transition in Esophageal Squamous Cell Carcinoma: In Vitro and Clinical Analyses of Cell Lines and Nomadic Kazakh Patients from Northwest Xinjiang, China

doi: 10.1371/journal.pone.0112300

Figure Lengend Snippet: Correlation between the expression of TGF-β1 protein and TGF-βRII/p-Smad2/3 proteins in ESCC.

Article Snippet: The blots were incubated with primary antibodies overnight at 4°C with rabbit anti-p-Smad2 (1∶1000, Cell Signaling Technology, Massachusetts, USA), anti-Smad7 (1∶200, Santa Cruz Biotechnology, California, USA), anti-E-cadherin (1∶500, Abcam, Cambridge, United Kingdom), mouse anti-vimentin (1∶100, Santa Cruz Biotechnology, California USA), or anti-N-cadherin antibodies (1∶1000, Abcam, Cambridge, United Kingdom)This procedure was followed by incubation with sheep anti-mouse (1∶20000) or anti-rabbit (1∶20000) HRP-labeled secondary antibodies for 2 h at room temperature.

Techniques: Expressing

Journal: Antioxidants

Article Title: Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis—Pharmacomodulation of Rosuvastatin

doi: 10.3390/antiox11122465

Figure Lengend Snippet: Protein expressions of EMT biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. ( A ) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( B ) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( C ) Protein expression of alpha smooth actin (α-SMA), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( D ) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( E ) Protein expression of E-cadherin (E-cad), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( F ) Protein expression of collagen type I (Coll-I), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( G ) Protein expression of laminin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( H ) Protein expression of elastin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). TGF-β = transforming growth factor-beta; EMT = epithelial mesenchymal transition; LDL = low-density lipoprotein.

Article Snippet: Briefly, primary antibodies against p-Smad2 (1:1000, Cell Signaling, Danvers, MA, USA), p-Smad2 (1:1000, Cell Signaling), Snail (1:1000, Cell Signaling), α-SMA (1:5000, Sigma, MA, USA), ferroptosis suppressor protein 1 (FSP1) (1:1000, Cell Signaling), E-cadherin (1:1000, Abcam, Cambridge, UK), Laminin (1:1000, Novus Biologicals, Centennial, CO, USA), Elastin (1:1000, Affinity Biosciences, Cincinnati, OH, USA), Collagen type I (1:5000, Sigma), fibronectin (1:1000, Abcam), transforming growth factor (TGF)-β1 (1:3000, Abcam), cleaved-Caspase3 (1:1000, Cell Signaling), cleaved-PARP (1:1000, Cell Signaling), mitochondrial Bax (1:1000, Abcam), NOX-1 (1:1000, Sigma), NOX-2 (1:1000, Sigma), oxidized protein (1:100, Millipore, Burlington, MA, USA), Vimentin (1:1000, Cell Signaling), matrix metalloproteinase (MMP)2 (1:1000, Cell Signaling), MMP9 (1:1000, Abcam), Actin (1:10,000, Chemicon, Tokyo, Japan), and COXIV (1:10,000, Abcam) were used.

Techniques: Expressing

Journal: Antioxidants

Article Title: Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis—Pharmacomodulation of Rosuvastatin

doi: 10.3390/antiox11122465

Figure Lengend Snippet: Protein expressions of EMT biomarkers in kidney parenchyma by day 42 after CKD induction. ( A ) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( B ) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( C ) Protein expression of α-SMA, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( D ) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡), p < 0.0001. ( E ) Protein expression of transforming growth factor (TGF)-1β, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( F ) Protein expression of vimentin, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( G ) Protein expression of E-cadherin, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Article Snippet: Briefly, primary antibodies against p-Smad2 (1:1000, Cell Signaling, Danvers, MA, USA), p-Smad2 (1:1000, Cell Signaling), Snail (1:1000, Cell Signaling), α-SMA (1:5000, Sigma, MA, USA), ferroptosis suppressor protein 1 (FSP1) (1:1000, Cell Signaling), E-cadherin (1:1000, Abcam, Cambridge, UK), Laminin (1:1000, Novus Biologicals, Centennial, CO, USA), Elastin (1:1000, Affinity Biosciences, Cincinnati, OH, USA), Collagen type I (1:5000, Sigma), fibronectin (1:1000, Abcam), transforming growth factor (TGF)-β1 (1:3000, Abcam), cleaved-Caspase3 (1:1000, Cell Signaling), cleaved-PARP (1:1000, Cell Signaling), mitochondrial Bax (1:1000, Abcam), NOX-1 (1:1000, Sigma), NOX-2 (1:1000, Sigma), oxidized protein (1:100, Millipore, Burlington, MA, USA), Vimentin (1:1000, Cell Signaling), matrix metalloproteinase (MMP)2 (1:1000, Cell Signaling), MMP9 (1:1000, Abcam), Actin (1:10,000, Chemicon, Tokyo, Japan), and COXIV (1:10,000, Abcam) were used.

Techniques: Expressing

Journal: Antioxidants

Article Title: Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis—Pharmacomodulation of Rosuvastatin

doi: 10.3390/antiox11122465

Figure Lengend Snippet: Protein expressions of EMT biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. ( A ) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( B ) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( C ) Protein expression of alpha smooth actin (α-SMA), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( D ) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( E ) Protein expression of E-cadherin (E-cad), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( F ) Protein expression of collagen type I (Coll-I), * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( G ) Protein expression of laminin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. ( H ) Protein expression of elastin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). TGF-β = transforming growth factor-beta; EMT = epithelial mesenchymal transition; LDL = low-density lipoprotein.

Article Snippet: Briefly, primary antibodies against p-Smad2 (1:1000, Cell Signaling, Danvers, MA, USA), p-Smad2 (1:1000, Cell Signaling), Snail (1:1000, Cell Signaling), α-SMA (1:5000, Sigma, MA, USA), ferroptosis suppressor protein 1 (FSP1) (1:1000, Cell Signaling), E-cadherin (1:1000, Abcam, Cambridge, UK), Laminin (1:1000, Novus Biologicals, Centennial, CO, USA), Elastin (1:1000, Affinity Biosciences, Cincinnati, OH, USA), Collagen type I (1:5000, Sigma), fibronectin (1:1000, Abcam), transforming growth factor (TGF)-β1 (1:3000, Abcam), cleaved-Caspase3 (1:1000, Cell Signaling), cleaved-PARP (1:1000, Cell Signaling), mitochondrial Bax (1:1000, Abcam), NOX-1 (1:1000, Sigma), NOX-2 (1:1000, Sigma), oxidized protein (1:100, Millipore, Burlington, MA, USA), Vimentin (1:1000, Cell Signaling), matrix metalloproteinase (MMP)2 (1:1000, Cell Signaling), MMP9 (1:1000, Abcam), Actin (1:10,000, Chemicon, Tokyo, Japan), and COXIV (1:10,000, Abcam) were used.

Techniques: Expressing

Journal: Antioxidants

Article Title: Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis—Pharmacomodulation of Rosuvastatin

doi: 10.3390/antiox11122465

Figure Lengend Snippet: Protein expressions of EMT biomarkers in kidney parenchyma by day 42 after CKD induction. ( A ) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( B ) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( C ) Protein expression of α-SMA, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( D ) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡), p < 0.0001. ( E ) Protein expression of transforming growth factor (TGF)-1β, * vs. other groups with different symbols (†, ‡), p < 0.0001. ( F ) Protein expression of vimentin, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. ( G ) Protein expression of E-cadherin, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Article Snippet: Briefly, primary antibodies against p-Smad2 (1:1000, Cell Signaling, Danvers, MA, USA), p-Smad2 (1:1000, Cell Signaling), Snail (1:1000, Cell Signaling), α-SMA (1:5000, Sigma, MA, USA), ferroptosis suppressor protein 1 (FSP1) (1:1000, Cell Signaling), E-cadherin (1:1000, Abcam, Cambridge, UK), Laminin (1:1000, Novus Biologicals, Centennial, CO, USA), Elastin (1:1000, Affinity Biosciences, Cincinnati, OH, USA), Collagen type I (1:5000, Sigma), fibronectin (1:1000, Abcam), transforming growth factor (TGF)-β1 (1:3000, Abcam), cleaved-Caspase3 (1:1000, Cell Signaling), cleaved-PARP (1:1000, Cell Signaling), mitochondrial Bax (1:1000, Abcam), NOX-1 (1:1000, Sigma), NOX-2 (1:1000, Sigma), oxidized protein (1:100, Millipore, Burlington, MA, USA), Vimentin (1:1000, Cell Signaling), matrix metalloproteinase (MMP)2 (1:1000, Cell Signaling), MMP9 (1:1000, Abcam), Actin (1:10,000, Chemicon, Tokyo, Japan), and COXIV (1:10,000, Abcam) were used.

Techniques: Expressing