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: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: ( A ) qPCR analyses reveal Tgfβ2 expression does not change in chick (blue circles; n=8) and quail (red squares; n=8) over time; however, in duck (yellow triangles; n=10), there is a decrease at HH37 and HH40; Tgfβ2 is higher in duck than chick or quail at HH31. ( B ) Acvrl1 does not change over time in chick; however, quail show an increase at HH37. There is a reduction in duck at HH40. Duck have higher Acvrl1 at HH34 compared to chick and quail. However, at HH37 and HH40, quail have more Acvrl1 compared to chick or duck. ( C ) Tgfβr2 does not change over time in chick or duck, but in quail, there is an induction at HH37. ( D ) Tgfβr3 does not change over time in chick, quail, or duck. ( E ) Smad3 in chick does not change until HH40 with a reduction. In quail and duck, Smad3 decreases at HH37 and continues to decrease for duck at HH40. Smad3 is higher in chick than quail at HH34 and is higher at HH37 compared to quail and duck. ( F ) Pai1 does not change over time in chick, but quail increase at HH37 and HH40, while duck decrease at the same time points. Duck Pai1 is higher at HH31 and HH34 compared to chick and quail, but at HH37 and HH40 quail it is significantly higher compared to chick and duck. ( G ) Mmp2 increases in chick and quail at HH37, whereas duck decrease at HH40. Chick have higher Mmp2 compared to duck at HH34 and HH37. Quail have higher Mmp2 compared to duck at HH37 and HH40. ( H ) Mmp9 increases in chick, quail, and duck at HH37. Quail have more Mmp9 compared to chick and duck at HH37 and HH40. p≤0.05 and * denotes significance from HH31 within each group, # denotes significance between quail and duck at same stage, † denotes significance between chick and quail, and ‡ 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), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Expressing

Journal: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: Protein levels for ( A ) phosphorylated (p) SMAD3 (50 kDa) and ( B ) MMP13 (54 kDa) in chick (n=9), quail (n=12), and duck (n=12) jaws from stages HH31 to HH40 with β-Actin (42 kDa) as a loading control. Dashed lines represent images taken from two separate fluorescent channels on the same gel, 680RD for β-Actin, and 800CW for pSMAD3 or MMP13. Figure 2—figure supplement 2—source data 1. Western blot images for phosphorylated SMAD and β-Actin. Figure 2—figure supplement 2—source data 2. Western blot images for MMP13 and β-Actin.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Control, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: ( A ) pSMAD3 protein levels in cells treated for 2 hr 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 1–24 hr. Chick Runx2 mRNA increases with rTGFβ1 treatment at 3 and 6 hr and at 24 hr, while duck Runx2 does not increase until 24 hr. In rTGFβ1 treated cells, duck have significantly lower Runx2 at every time point compared to chick (n=8). ( C ) Chick Mmp13 mRNA increases with rTGFβ1 treatment at 6 hr and at 24 hr, while duck Runx2 does not increase until 24 hr. In rTGFβ1 treated cells, duck have lower Mmp13 at every time point compared to chick (n=8). ( D ) MMP13 protein in cells treated for 24 hr with rTGFβ1 shows an induction in chick but no response in duck (n=8). ( E ) RUNX2 protein levels in chick cells treated for 24 hr 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 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 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, and ‡ 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), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Control

Journal: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: Protein levels for ( A ) phosphorylated (p) SMAD3 (50 kDa) and ( B ) MMP13 (54 kDa) in chick (DF-1) and duck (CCL-141) cells treated with 5 ng/ml rTGFβ1 for 2 hr. β-Actin (42 kDa) is used as a loading control (n=8). Figure 3—figure supplement 1—source data 1. Western blot images for pSMAD and β-Actin. Figure 3—figure supplement 1—source data 2. Western blot images for MMP13 and β-Actin.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Control, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: Protein levels for ( A ) RUNX2 (55 kDa) and ( B ) MMP13 (54 kDa) in chick cells treated for 24 hr with rTGFβ1, TGFβR1 inhibitor, a combination of both rTGFβ1 and TGFβR1 inhibitor, SMAD3 inhibitor, and a combination of both rTGFβ1 and SMAD3 inhibitor (n=8). β-Actin (42 kDa) is used as a loading control. Figure 3—figure supplement 3—source data 1. Western blot images for RUNX2 and β-Actin. Figure 3—figure supplement 3—source data 2. Western blot images for MMP13 and β-Actin.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Control, Western Blot

Journal: eLife

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

doi: 10.7554/eLife.66005

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. ( G ) In ovo electroporations of the pPIDNB- Mmp13 OE construct were performed at HH8.5 and embryos were allowed to develop until HH35 when they were treated with a single dose of dox. Electroporation efficiency and extent of OE were evaluated during embryo collection at HH40 by detecting RFP (red). Microcomputed tomography (µCT) analysis of duck specimens electroporated with ( H ) empty vector or ( I ) Mmp13 OE construct. A 3D model was annotated with landmarks from which ( J ) lower jaw distances were calculated. Embryos overexpressing Mmp13 showed significantly shorter jaws compared to empty vector controls. * denotes significance from control within each group p≤0.05, ** denotes significance from control within each group p≤0.01, and *** denotes significance from control within each group p≤0.001. Figure 5—source data 1. Effects of TGFβR1, SMAD3, and MMP13 inhibition on TRAP staining in quail embryos. Figure 5—source data 2. Effects of MMP13 overexpression on jaw length in duck embryos as visualized by µCT.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Control, Staining, Comparison, In Ovo, Construct, Electroporation, Tomography, Plasmid Preparation, Inhibition, Over Expression

Journal: eLife

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

doi: 10.7554/eLife.66005

Figure Lengend Snippet: Graphical representation of the binding profiles of the position weight matrices taken from the JASPAR 2020 database for ( A ) RUNX2, ( B ) SMAD2/3 heterodimer, ( C ) SMAD3, and ( D ) SMAD4. The relative sizes of the letters indicate their frequency in the sequences, whereas the height of the letters shows the information content of the position.

Article Snippet: Membranes were probed with 1:1000 rabbit anti-human pSer423/pSer425 SMAD3 antibody (NBP1-77836, Novus Biologicals, Littleton, CO), 1:1000 rabbit anti-human SMAD3 antibody (NB100-56479, Novus Biologicals, Littleton, CO), 1 μg/ml rabbit anti-chick MMP13 custom-made primary antibody (GenScript, Piscataway, NJ; ), 1:1000 rabbit anti-human MMP2 antibody (NB200-193, Novus Biologicals, Littleton, CO), 1:4000 mouse anti-human β-actin antibody (NB600-501, Novus Biologicals, Littleton, CO), 1:15,000 goat anti-rabbit IRDye 800CW (925–32211, LI-COR, Lincoln, NE), and 1:15,000 donkey anti-mouse IRDye 680RD antibody ( ; 925–68072, LI-COR, Lincoln, NE).

Techniques: Binding 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: 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: Oncotarget

Article Title: Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5

doi: 10.18632/oncotarget.9600

Figure Lengend Snippet: A. - E. Immunoblot analysis of C-terminally phosphorylated Smad3 (p-Smad3C) in Panc-1 A . Colo357 B . IMIM-PC1 C . and HaCaT D . cells treated with transfection agent alone (−) or transiently transfected with 50 nM of control (Co) siRNA, or siRNA specific to PAR2, PAR1, or ALK5 as indicated. Forty-eight h after the start of transfection, cells were stimulated or not with TGF-β1 for 1 h. In A-D, blots were stripped and reprobed with antibodies to Smad3 and β-actin to control for equal loading. E . Immunoblot analysis of phospho-p38 (p-p38), and p38 as loading control, in Panc-1 cells transfected with the indicated siRNAs and treated or not (−) with TGF-β1 for various times as indicated. In each panel, a representative blot from at least three independent experiments is shown. Successful knockdown of PAR2, ALK5 and PAR1 expression was verified by qPCR (not shown). M, molecular weight marker.

Article Snippet: The following antibodies were used: ALK5 (V-22), HSP90α/β (H-114, sc-7947), and PAR2 (SAM11, sc-13504, Fluorescein isothiocyanate (FITC)-conjugated) (Santa Cruz Biotechnology, Heidelberg, Germany), Caveolin (BD Transduction Laboratories, Heidelberg, Germany, #610059), phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-Smad2(Ser465/467) (all from Cell Signaling Technology, Frankfurt, Germany, #4370, #3104 and #2276, respectively), phospho-Smad3(Ser423/425) (R&D Systems, Wiesbaden, Germany, #ab3226), Smad2 (Epitomics, Burlingame, CA, #1736-1), Smad3 (Abcam, Cambridge, UK, #ab40854), TGF-β Receptor II (Cell Signaling Technology, #11888), β-actin (Sigma, Deisenhofen, Germany).

Techniques: Western Blot, Transfection, Expressing, Molecular Weight, Marker