Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Validation of the differential expression of miR-130a-3p in the livers of mice and patients. (a ) miR-130a-3p expression was decreased, and the expression of TGFBR1, TGFBR2, Col-1, and Col-4 was increased in the liver tissues of the MCD-fed mice. TGFBR1, TGFBR2, Col-1, and Col-4 expression was detected by IHC ( × 200 magnification), and miR-130a-3p expression was detected using an ISH ( × 400 magnification) assay. Positive staining is indicated by a brown color. (b) Effects of the MCD diet on serum ALT and AST levels. Values represent the mean±S.D., *** P <0.001 compared with control. (c) Hepatic mRNA and (d) protein expression of TGFBR1, TGFBR2, Smad2, Smad3, Col-1, and Col-4 were upregulated in the MCD-fed mice compared with the control mice. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control. (e) Histopathological changes were evaluated with hematoxylin and eosin staining, and Masson’s trichrome staining. miR-130a-3p expression was decreased, and TGFBR1, TGFBR2, Col-1, and Col-4 levels were increased in the liver tissues of patients with NASH-related liver fibrosis. The expression of miR-130a-3p was detected by ISH ( × 400 magnification), and TGFBR1, TGFBR2, Col-1, and Col-4 were detected using an IHC ( × 200 magnification) assay. Positive staining is indicated by a brown color

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Biomarker Discovery, Quantitative Proteomics, Expressing, Immunohistochemistry, In Situ Hybridization, Staining, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: miR-130a-3p expression was downregulated and regulated the downstream expression of genes of the TGF-β/SMAD signaling pathway in activated HSCs. ( a ) miR-130a-3p expression was examined by real-time qRT-PCR during HSC activation. Values represent the mean±S.D., (* P <0.05, ** P <0.01). ( b ) The hepatic mRNA and ( c ) protein expression of TGF- β 1 , α -SMA, Smad2, and Smad3 were increased during the process of HSC activation. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( d ) HSC-T6 cells were transfected with miR-130a-3p mimics or mimics control for 48 h. The mRNA and protein ( e ) levels of TGF- β 1 , Smad2, and Smad3 were analyzed by qRT-PCR and western blotting, respectively. Mimics control is the negative control of mimics, and control is the blank control in this group. The result showed that there was no significant difference between control and mimics control. β -actin was used as a loading control. Values represent the mean±S.D., ** P <0.01, *** P <0.001 compared with control. ( f ) The mRNA and protein ( g ) levels of MMP-2, MMP-9, Col-1, and Col-4 were analyzed by qRT-PCR and western blotting, respectively. β -actin was used as a loading control. Values represent the mean±S.D., * P <0.05, ** P <0.01, *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Control, Transfection, Western Blot, Negative Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Knockdown of either TGFBR1 or TGFBR2 inhibited the TGF- β / SMAD signaling pathway and reduced HSC activation. ( a ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( b ) of TGFBR1 and TGFBR2. β-actin was used as a loading control. Values represent the mean ±SD, *** P < 0.001 compared with control. ( c ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein expression levels ( d ) of α -SMA, Smad2, and Smad3. β-actin was used as a loading control. Values represent the mean ±SD, * P <0.05, ** P <0.01, *** P <0.001 compared with control. ( e ) Knockdown of TGFBR1 and/or TGFBR2 inhibited the mRNA and protein levels ( f ) of MMP-2, MMP-9, Col-1, and Col-4. β -actin was used as a loading control. Values represent the mean±S.D., *** P <0.001 compared with control

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Knockdown, Activation Assay, Expressing, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Overexpression of TGFBR1 and TGFBR2 rescued miR-130a-3p-impaired proliferation and cell growth in HSCs. The HSC-T6 cells were transduced with the miR-130a-3p mimics and the TGFBR1-expressing and TGFBR2-expressing vectors. The rescue efficiency of TGFBR1 or TGFBR2 in the HSC-T6 cells was confirmed by ( a ) RT-PCR and ( b ) western blotting. β -actin was used as a loading control. Values represent the mean±S.D. (*** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the (c) mRNA and ( d ) protein expression of Smad2 and Smad3. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). Overexpression of TGFBR1 and TGFBR2 rescued the ( e ) mRNA and ( f ) protein expression of Col-1 and Col-4. β -actin was used as a loading control. Values represent the mean±S.D. (* P <0.05, ** P <0.01, *** P <0.001). ( g ) A schematic diagram highlighting the regulation of HSCs by miR-130a-3p during the genesis and resolution of liver fibrosis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Over Expression, Transduction, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MiR-130a-3p attenuates activation and induces apoptosis of hepatic stellate cells in nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2

doi: 10.1038/cddis.2017.10

Figure Lengend Snippet: Primers for quantitative real-time PCR analysis

Article Snippet: After being blocked for 60 min with buffer containing 0.1% Tween-20 and 5% milk, the membranes were incubated overnight at 4 °C with primary antibodies against α -SMA, Col-1, Col-4 (Bioss, Beijing, China), Smad2, Smad3 (Novus Biologicals, Novus Biologicals, Littleton, CO, USA), TGFBR1 (Abcam), TGFBR2 (Novus Biologicals), MMP-2, MMP-9 (ProteinTech Group, Chicago, IL, USA), TGF- β 1 , caspase-3, caspase-9, and PARP1 (Novus Biologicals, Littleton, CO, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution

doi: 10.1101/2020.12.23.424223

Figure Lengend Snippet: (A) pSMAD3 protein levels in cells treated for 2 hours with 5 ng/ml rTGFβ1 (dark gray) in chick (blue) and duck (yellow) cells show a significant induction in chick (n = 8). (B) Chick and duck cells treated with rTGFβ1 for 1 to 24 hours. Chick Runx2 mRNA increases 3-fold with rTGFβ1 treatment at 3 and 6 hours and 8-fold at 24 hours, while duck Runx2 does not increase until 24 hours. In rTGFβ1 treated cells, duck have significantly lower Runx2 at every time point compared to chick (n = 8). (C) Chick Mmp13 mRNA increases 4.5-fold with rTGFβ1 treatment at 6 hours and 10-fold at 24 hours, while duck Runx2 does not increase until 24 hours. In rTGFβ1 treated cells, duck has lower Mmp13 at every time point compared to chick (n = 8). (D) MMP13 protein in cells treated for 24 hours with rTGFβ1 show a 1.5-fold induction in chick but no response in duck (n = 8). (E) RUNX2 protein levels in chick cells treated for 24 hours with rTGFβ1 (dark gray), TGFβR1 inhibitor (medium gray), a combination of both rTGFβ1 and TGFβR1 inhibitor (black), SMAD3 inhibitor (white), and a combination of both rTGFβ1 and SMAD3 inhibitor (light gray). RUNX2 protein increases 2-fold with rTGFβ1 but when rTGFβ1 is combined with either a TGFβr1 or a SMAD3 inhibitor, there is a significant decrease compared to rTGFβ1 alone (n = 12). (F) MMP13 protein increases 2.3-fold with rTGFβ1 treatment but when rTGFβ1 is combined with either a TGFβR1 or a SMAD3 inhibitor, there is a significant decrease compared to rTGFβ1 alone (n = 12). * denotes significance from control p < 0.05, ** denotes significance from control p < 0.01, *** denotes significance from control p < 0.001, # denotes significance from rTGFβ1, † denotes significance between chick and quail, ‡ denotes significance between chick and duck.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO, USA), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO, USA), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ, USA; Supplemental Table 2), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO, USA), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO, USA), 1:15000 goat anti-rabbit IRDye 800CW (925-32211, LI-COR, Lincoln, NE, USA), and 1:15000 donkey anti-mouse IRDye 680RD antibody (925-68072, LI-COR, Lincoln, NE, USA).

Techniques: Control

Journal: bioRxiv

Article Title: Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution

doi: 10.1101/2020.12.23.424223

Figure Lengend Snippet: Quail lower jaws harvested at HH35 and placed in culture with control beads (left side) and treatment beads (right side) soaked in (A) TGFβR1 inhibitor (TGFβi, n = 5), (B) SMAD3 inhibitor (SMAD3i, n = 5), and (C) MMP13 inhibitor (MMP13i, n = 5). The effects of inhibitor treatments can be seen on TRAP staining (red) after 5 days of culture (red dashed lines). Quantification of the % area of TRAP-positive staining for (D) TGFβR1 inhibitor (medium gray), (E) SMAD3 inhibitor (white), and (F) MMP13 inhibitor (dark gray) in comparison to the contralateral control side. * denotes significance from control within each group p < 0.05, ** denotes significance from control within each group p < 0.01, *** denotes significance from control within each group p < 0.001.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO, USA), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO, USA), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ, USA; Supplemental Table 2), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO, USA), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO, USA), 1:15000 goat anti-rabbit IRDye 800CW (925-32211, LI-COR, Lincoln, NE, USA), and 1:15000 donkey anti-mouse IRDye 680RD antibody (925-68072, LI-COR, Lincoln, NE, USA).

Techniques: Control, Staining, Comparison

Journal: Diabetes

Article Title: Blockade of Endothelial-Mesenchymal Transition by a Smad3 Inhibitor Delays the Early Development of Streptozotocin-Induced Diabetic Nephropathy

doi: 10.2337/db09-1631

Figure Lengend Snippet: AGE-induced activation of Smad3 was RAGE mediated in MMECs. MMECs were cultured in the presence of AGEs and BSA for 15–120 min or with different concentrations of AGEs for 30 min. Immunoprecipitation (IP) and Western blotting (WB) demonstrated the time course ( A ) and dose response ( B ) of Smad3 phosphorylation (p-Smad3) and total Smad3 levels in MMECs. C : MMECs were pretreated with control siRNA (CTL siRNA) or RAGE siRNA for 2 days and then cultured in the presence of BSA or AGEs for 30 min. Immunoprecipitation/Western blotting demonstrated RAGE, p-Smad3, and total Smad3 in MMECs. D : MMECs were pretreated with goat anti-RAGE neutralizing antibody or goat IgG for 30 min and then cultured in the presence of AGEs. Upper panel : Immunoprecipitation/Western blotting demonstrated Smad3 phosphorylation and total Smad3 in MMECs. Lower panel : quantitation of arbitrary ratio of Smad3 phosphorylation/Smad3 in three independent experiments. a, vs. BSA-treated group, P < 0.05; b, vs. AGEs or AGEs plus goat IgG, P < 0.05. E : MMECs were pretreated with mouse anti–TGF-β1 neutralizing antibody or mouse IgG for 30 min and then cultured in the presence of AGEs for 30 min. Immunoprecipitation/Western blotting demonstrated Smad3 Smad3 phosphorylation and total Smad3 in MMECs. F : MMECs were pretreated with control siRNA or TGF-β receptor 1 siRNA for 2 days and then cultured in the presence of AGEs for 30 min. Immunoprecipitation/Western blotting demonstrated TGF-β receptor 1, GAPDH, p-Smad3, and total Smad3 in MMECs.

Article Snippet: The following antibodies were used for immunofluorescence studies: rat anti-CD31(BD Biosciences, San Diego, CA), rabbit anti–Von Willebrand factor (vWF) (Dako, Glostrup, Denmark), mouse anti–α-SMA conjugated with cyanine three (Sigma-Aldrich), rat anti–VE-cadherin (eBioscience, San Diego, CA), rabbit anti–phosphorylated Smad3 (Novus Biologicals, Littleton, CO), rabbit anti-fibronectin (Sigma-Aldrich), goat anti–collagen IV, goat anti–rabbit Alexa Fluor 555 conjugate, goat anti–rat Alexa Fluor 647 conjugate, and chicken anti–goat 647 conjugate (Invitrogen).

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

Journal: Diabetes

Article Title: Blockade of Endothelial-Mesenchymal Transition by a Smad3 Inhibitor Delays the Early Development of Streptozotocin-Induced Diabetic Nephropathy

doi: 10.2337/db09-1631

Figure Lengend Snippet: SIS3 inhibited AGE-induced activation of Smad3 and EndoMT in MMECs. MMECs were pretreated with 1 μmol/l SIS3 or DMSO for 30 min and then cultured in the presence of 25 μg/ml AGEs for 30 min or 7 days. Immunoprecipitation/Western blotting demonstrated Smad3 phosphorylation (p-Smad3), Smad3, p-Smad2, and Smad2 at 30 min after AGEs stimulation in the presence of SIS3 ( A ). Confocal microscopy demonstrated the expression of VE-cadherin (green), α-SMA (red), and DAPI (blue) 7 days after incubation with BSA ( C ), AGEs plus DMSO ( D ), and AGEs plus SIS3 ( E ) in MMECs. Arrows indicate VE-Cadherin + /α-SMA + cells. B : Quantitation of percentages of α-SMA– and VE-cadherin–positive cells in total DAPI-positive cells. a, vs. BSA-treated group or AGEs plus SIS3–treated group, P < 0.05. Original magnification 600×. F and G : MMECs were pretreated with control siRNA (CTL siRNA), Smad2 siRNA, or Smad3 siRNA for 2 days and then cultured in the presence of AGEs or BSA for 24 h. Western blotting demonstrated Smad2, Smad3, and GAPDH in MMECs ( F ), and real-time PCR showed α-SMA mRNA levels in MMECs ( G ). b, vs. BSA, P < 0.05. c, vs. BSA, P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: The following antibodies were used for immunofluorescence studies: rat anti-CD31(BD Biosciences, San Diego, CA), rabbit anti–Von Willebrand factor (vWF) (Dako, Glostrup, Denmark), mouse anti–α-SMA conjugated with cyanine three (Sigma-Aldrich), rat anti–VE-cadherin (eBioscience, San Diego, CA), rabbit anti–phosphorylated Smad3 (Novus Biologicals, Littleton, CO), rabbit anti-fibronectin (Sigma-Aldrich), goat anti–collagen IV, goat anti–rabbit Alexa Fluor 555 conjugate, goat anti–rat Alexa Fluor 647 conjugate, and chicken anti–goat 647 conjugate (Invitrogen).

Techniques: Activation Assay, Cell Culture, Immunoprecipitation, Western Blot, Phospho-proteomics, Confocal Microscopy, Expressing, Incubation, Quantitation Assay, Control, Real-time Polymerase Chain Reaction

Journal: Diabetes

Article Title: Blockade of Endothelial-Mesenchymal Transition by a Smad3 Inhibitor Delays the Early Development of Streptozotocin-Induced Diabetic Nephropathy

doi: 10.2337/db09-1631

Figure Lengend Snippet: SIS3 inhibited Smad3 activation in STZ-induced diabetic nephropathy. Immunoprecipitation/Western blotting demonstrated the time course of phosphorylated Smad3 (p-Smad3) and total levels of Smad3 in STZ-induced diabetic mouse kidneys and normal saline (NS)-treated kidneys ( A ) and p-Smad3 and total Smad3 in normal saline-treated, STZ-injected nondiabetic (STZ+/DM−) and STZ-injected diabetic (STZ+/DM+) mouse kidneys ( B ). C : One month after the onset of STZ-induced diabetes or normal saline treatment, diabetic mice were given intraperitoneal injection of vehicle and different dosages of SIS3. Immunoprecipitation/Western blotting demonstrated p-Smad3, total Smad3, p-Smad2, and total Smad2 in kidneys of mice with different treatments. D : Immunoprecipitation/Western blotting demonstrated p-Smad3 and total Smad3 in normal saline-treated mouse kidneys, STZ-induced diabetic nephropathy plus vehicle-treated mouse kidneys, and STZ-induced diabetic nephropathy plus SIS3-treated (2.5 μg · g −1 · day −1 ) mouse kidneys. Confocal microscopy demonstrated CD31 (green), p-Smad3 (red), and DAPI (blue) staining in 1-month normal saline-treated kidney ( E ), 1-month STZ-induced diabetic kidney ( F ), 3-month STZ-induced diabetic nephropathy plus vehicle-treated mouse kidney ( H ), and 3-month STZ-induced diabetic nephropathy plus SIS3-treated mouse kidney ( I ). G : Quantitation of percentage of phosporylated Smad3–positive cells in total CD31-positive cells in 1-month normal saline-treated and STZ-injected diabetic kidneys. J : Quantitation of percentage of phosphorylated Smad3–positive cells in total CD31-positive cells in 3-month normal saline-treated and STZ-induced diabetic nephropathy plus vehicle-treated and STZ-induced diabetic nephropathy plus SIS3 treated kidneys. a, vs. normal saline, P < 0.05; b, vs. STZ-induced diabetic nephropathy plus vehicle-treated kidneys, P < 0.05. Original magnification: C , D , F , and G , 600×. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: The following antibodies were used for immunofluorescence studies: rat anti-CD31(BD Biosciences, San Diego, CA), rabbit anti–Von Willebrand factor (vWF) (Dako, Glostrup, Denmark), mouse anti–α-SMA conjugated with cyanine three (Sigma-Aldrich), rat anti–VE-cadherin (eBioscience, San Diego, CA), rabbit anti–phosphorylated Smad3 (Novus Biologicals, Littleton, CO), rabbit anti-fibronectin (Sigma-Aldrich), goat anti–collagen IV, goat anti–rabbit Alexa Fluor 555 conjugate, goat anti–rat Alexa Fluor 647 conjugate, and chicken anti–goat 647 conjugate (Invitrogen).

Techniques: Activation Assay, Immunoprecipitation, Western Blot, Saline, Injection, Confocal Microscopy, Staining, Quantitation Assay

Journal: BMC cancer

Article Title: Valproic acid sensitizes metformin-resistant human renal cell carcinoma cells by upregulating H3 acetylation and EMT reversal.

doi: 10.1186/s12885-018-4344-3

Figure Lengend Snippet: Fig. 8 Tentative roles of aH3, pSMAD3, pAMPK and pAKT in RCC cells and metformin resistant RCC cells. Long-term application of metformin induces resistance in RCC and this resistance mainly based on the loss of sensitivities of AMPK/ AKT and TGF- β/SMAD3 pathways. However, the addition of VPA can counteract the resistance through increasing the sensitivities of pAKT by upregulation of aH3 and reversing the EMT process by inhibiting pSMAD3/SMAD4

Article Snippet: The blots were blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) and 5% milk or 5% BSA for 1 h at room temperature, then incubated with primary antibodies against pAMPK, p-AKT, total AKT, pSMAD3, SMAD3, SMAD4, aH3, H3 (Abcam) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C.

Techniques:

Journal: Cellular & molecular biology letters

Article Title: A therapeutic target for CKD: activin A facilitates TGFβ1 profibrotic signaling.

doi: 10.1186/s11658-023-00424-1

Figure Lengend Snippet: Fig. 1 Activin inhibition attenuates TGFβ1-induced fibrotic responses and Smad3 activation in MC. Activin inhibition with follistatin (FST) decreases TGFβ1-induced: a FN, αSMA and CTGF upregulation at 48 h (n = 5), b Smad3 phosphorylation (pSmad3) at 24 h (n = 5), c Smad3 nuclear translocation as assessed using eGFP-Smad3 (n = 3; 25–30 cells quantified per treatment group) at 24 h, and d Smad3 transcriptional activity at 24 h (n = 8). e Time course experiments show increases in pSmad3 occur earlier (30–60 min) with TGFβ1 (n = 4) compared with actA (n = 4) or actB (n = 3) (18–48 h). *, **, ***, ****P < 0.05, 0.01, 0.001, 0.0001; one-way ANOVA with Tukey’s multiple comparisons post hoc test

Article Snippet: Primary antibodies were: fibronectin (BD Transduction; 610078), αSMA (Pierce; MA1-06110), CTGF (Sigma; AMAB91366), Smad3 (Abcam; ab40854), pSmad3 (Novus; NBP1-77836), pSmad2 (Cell Signaling; 3108), Smad2/3 (Cell Signaling; 8685) MRTF-A (Abcam; ab49311), GAPDH (Millipore; CB1001), TRI (Abcam; ab31013), TRII (Abcam; ab78419), ALK4 (Abcam, ab109300) PDGFR (Cedarlane; 1469-1), tubulin (Invitrogen; 32-2700) and lamin B (Santa Cruz; ac-6217).

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Translocation Assay, Activity Assay

Journal: Cellular & molecular biology letters

Article Title: A therapeutic target for CKD: activin A facilitates TGFβ1 profibrotic signaling.

doi: 10.1186/s11658-023-00424-1

Figure Lengend Snippet: Fig. 6 Activin A neutralization inhibits renal fibrosis in TGFβ1-overexpressing mice. a TGFβ1 transcript is increased in mice genetically engineered to overexpress TGFβ1 (HH) compared with wild-type mice (WT) (n = 6–7, *p ≤ 0.05). b Serum actA is elevated in wild-type and HH mice after UUO. This is decreased by treatment with a neutralizing actA antibody (anti-actA) in HH mice. c Renal actA is increased after UUO, with a greater induction in HH mice. Both are attenuated by actA neutralization. Boxed areas are shown at higher magnification immediately below. ActA increases are seen particularly in tubular epithelial cells. d Renal αSMA, fibronectin (FN), pSmad3 and MRTF-A are increased after UUO and this is augmented in HH kidneys. Expression of all is attenuated by actA neutralization in both WT and HH kidneys. (n = 6–9) *, **, ***, ****P < 0.05, 0.01, 0.001, 0.0001; one-way ANOVA with Tukey’s multiple comparisons post hoc test where there are > 2 groups; t-test for 2 groups

Article Snippet: Primary antibodies were: fibronectin (BD Transduction; 610078), αSMA (Pierce; MA1-06110), CTGF (Sigma; AMAB91366), Smad3 (Abcam; ab40854), pSmad3 (Novus; NBP1-77836), pSmad2 (Cell Signaling; 3108), Smad2/3 (Cell Signaling; 8685) MRTF-A (Abcam; ab49311), GAPDH (Millipore; CB1001), TRI (Abcam; ab31013), TRII (Abcam; ab78419), ALK4 (Abcam, ab109300) PDGFR (Cedarlane; 1469-1), tubulin (Invitrogen; 32-2700) and lamin B (Santa Cruz; ac-6217).

Techniques: Neutralization, Expressing

Journal: Molecular Vision

Article Title: Notch prevents transforming growth factor-beta-assisted epithelial–mesenchymal transition in cultured limbal progenitor cells through the induction of Smad7

doi:

Figure Lengend Snippet: Suppressing Smad7 protein expression with Notch inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester facilitates epithelial–mesenchymal transition and growth arrest induced by transforming growth factor-beta. A : Western blot analysis of Smad 2/3 phosphorylation. Left planes: Transforming growth factor-beta (TGFβ1) time-dependently induces Smad2/3 phosphorylation in P1 cells. Right planes: P1 cells were treated with 10 ng/ml TGFβ1 for 30 and 40 min as positive control. P0 and P1 cells were pretreated with 10 µM N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or dimethyl sulfoxide (DMSO) vehicle for 48 h and then stimulated with TGFβ1 for 30 and 40 min, respectively. Cells were harvested and subjected to western blot analysis with phosphospecific antibodies of Smad2 and Smad3. The total protein levels of Smad2 and Smad3 were estimated by reprobing the membranes with total Smad2 and Smad3 antibody shown in the panels below. B : P0 LSCs pretreated with DAPT facilitates mesenchymal marker expression induced by TGFβ1. Representative blots (left panels) and densitometric analysis with standard deviation (SD; right columns) of three independent experiments are shown. *p<0.05 versus DMSO/TGFβ1-treated cells. C : P0 LSCs pretreated with DAPT facilitates growth arrest induced by TGFβ1. Cells were pretreated with DMSO or DAPT for 48 h and then treated with TGFβ1 for further 24 h. Subsequently, total RNA was extracted for determining Ki67 mRNA with quantitative real-time PCR (qPCR) assay as described in . Data represent three independent experiments. *p<0.002 versus DMSO/TGFβ1-treated cells.

Article Snippet: Samples were probed with anti-∆Np63α body (fold dilution, 1:1,000; BioLegend, San Diego, CA), anti-Bmi-1 antibody (1:1,000; Millipore, Bedford, MA), anti-ABCG2 antibody (1:1,000; Abcam, Cambridge, MA), anti-Smad7 antibody (1:1,000; GeneTex, San Antonio, Tex), anti-Hes1 antibody (1:1,000; GeneTex), anti-NICD antibody (1:1,000; Cell Signaling Technology Inc., Beverly, MA), anti-phospho-Smad2 antibody (1:1,000; S465/S467, Upstate Biotechnology, Lake Placid, NY), anti-phospho-Smad3 antibody (1:1,000; S423/S425, R&D Systems, Minneapolis, MN), anti-Smad2 antibody (1:1,000; Abcam), Smad3 (1:1,000; Zymed Laboratories, San Francisco, CA), anti-TGFβ1 antibody (1:1,000; GeneTex), anti-TGFβRI antibody (1:1,000; GeneTex), or anti-TGFβRII antibody (1:1,000; GeneTex). β-actin (1:1,0000; Sigma-Aldrich) were used to verify equal loading of protein.

Techniques: Expressing, Western Blot, Phospho-proteomics, Positive Control, Marker, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: Fibrogenic gene and EMT-related gene expression levels were induced in the BSA-injected diabetic mouse kidney; TENE treatment suppressed these gene expression levels. ( a ) Microarray analysis of the kidney samples. Heat map analysis of the gene expression. BSA injection (particularly in the diabetic mice) induced genes, such as DPP-4, TGF-β/smad3 signaling, CAV1, integrin β1 and the EMT program; TENE treatment suppressed these alterations ( n = 2 mice per group, and the average value is shown in the figure). Red indicates high and green indicates low relative expression levels. ( b – p ) qPCR analysis of the expression of the indicated genes in the kidney of mice in each group ( n = 7 mice per group). Gene expression was normalized to the control mice value. * P < 0.05, ** P < 0.01. Data are presented as mean ± s.e.m. ( q ) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 7 per group were analyzed.

Article Snippet: A rabbit polyclonal anti-phospho smad3 (s423 and s425, 1:500) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA).

Techniques: Expressing, Injection, Microarray, Western Blot

Journal: Scientific Reports

Article Title: Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice

doi: 10.1038/s41598-019-43730-5

Figure Lengend Snippet: TENE treatment suppressed the crosstalk among DPP-4, integrin β1 and CAV1 via inhibition of TGF-β/smad3 signaling pathway in vitro . Duolink in situ analysis of (a-c) DPP-4/integrin β1, ( d – f ) DPP-4/CAV1 and ( g – i ) integrin β1/CAV1 in HK-2 cells with or without TGF-β1 (10 ng/ml) was performed by confocal microscopy (×1260). Scale bar: 50 μm in each panel. ( j ) Representative western blot analysis. As a densitometric analysis, each protein level was normalized with actin. n = 6 per group were analyzed. ( k – n ) Duolink in situ analysis of integrin β1/CAV1 in DPP-4 overexpressed HK-2 cells with or without TENE and SIS3. ( o ) Immunoprecipitation analysis revealed TGF-β treatment increased crosstalk among DPP-4, integrin β1 (ITGβ1) and CAV1. ( p ) Immunoprecipitation assay revealed TGF-β neutralization suppressed crosstalk among DPP-4, integrin β1 and CAV1 induced by DPP-4 overexpression.

Article Snippet: A rabbit polyclonal anti-phospho smad3 (s423 and s425, 1:500) antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA).

Techniques: Inhibition, In Vitro, In Situ, Confocal Microscopy, Western Blot, Immunoprecipitation, Neutralization, Over Expression

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: The Traditional Chinese Medicine Formula FTZ Protects against Cardiac Fibrosis by Suppressing the TGF β 1-Smad2/3 Pathway

doi: 10.1155/2022/5642307

Figure Lengend Snippet: FTZ improves cardiac fibrosis by inhibiting the TGF β 1-Smad2/3 pathway. (a) mRNA expression of TGF β 1 was determined using RT-qPCR. n = 5 per group. (b–e) Protein expression of TGF β 1, p-smad2, smad2, p-smad3, and smad3 were determined using western blotting. n = 5 per group. Data are presented as the mean ± SEM. ∗∗ P < 0.01 versus the control group and ## P < 0.01 versus the Ang-II group. (f) and (g) TGF β 1 expression of consecutive sections of cardiac specimens assessed using immunohistochemical staining. Scale bar: 50 μ m. n = 3 per group. Data are presented as the mean ± SEM. ∗∗ P < 0.01 versus the sham group and ## P < 0.01 versus the TAC group.

Article Snippet: The antibodies included p-smad2 (AbSci, 1:500), smad2 (Proteintech, 1:1,000), p-smad3 (AbSci, 1:500), smad3 (Proteintech, 1:1,000), TGF β 1 (Proteintech, 1:1,000), Col1A2 (Proteintech, 1:2,000), Col3 (Proteintech, 1:500), α -SMA (Proteintech, 1:20,000), and β -actin (Proteintech, 1:2,000).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining