Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Endothelial cell tubule formation assay. BME cells (3 × 105/ml) were seeded onto type I collagen gels in medium supplemented with 10% calf serum. On the following day, the medium was aspirated and replaced with conditioned serum-free medium derived from cocultures of G8 myoblast monolayers and TA3wt (A) TA3sCD44 (B), TA3sCD44/MMP-9-CD44fp (C), or TA3sCD44/MMP-9v5 (D) cells. A total of 30 μg/ml of pan specific anti-TGF-β (E) or anti-bFGF (F) antibody were added to the TA3wt/G8-conditioned coculture medium prior to use in the assay. Tubules are indicated by arrows. Bar, 140 μm.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Tube Formation Assay, Derivative Assay

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: CD44-anchored MMP-9 activates TGF-β. (A) The ability of TA3 transfectant-G8 myoblast coculture media to stimulate TMLC luciferase activity is shown. Conditioned coculture media tested are indicated. (B) Pan-specific TGF-β antibody (30 μg/ml) or specific antibodies against TGF-β1 (100 ng/ml), TGF-β2(100ng/ml), or TGF-β3(100 ng/ml) were used to determine the activity of TGF-β isoforms in the coculture media. One unit of luciferase activity corresponds to the activity produced by 5 pg of purified human TGF-β1 (R & D).

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Activity Assay, Produced, Purification

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: TGF-β is activated by purified MMP-9. (A) TMLC luciferase activity induced by concentrated COS cell supernatants containing the indicated latent TGF-β isoforms following incubation with the indicated purified AMPA-activated MMPs. Comparable amounts of latent TGF-β1, TGF-β2, and TGF-β3 were present in the supernatants as assessed by both Western blot analysis and TMLC-luciferase induction following heat treatment (80°C for 5min) (B) TMLC luciferase activity induced by affinity-purified TGF-β2 following incubation with the indicated purified activated MMPs. Activity is expressed in relative light units (RLU), in which 800 RLU corresponds to the luciferase activity generated by 1 pg of purified human TGF-β1 (R & D). (C) TGF-β is proteolytically cleaved by MMP-9 and MMP-2. Purified v5-tagged TGF-β2 (lane 1) was incubated with protein-A Sepharose-bound, AMPA-activated MMP-9 (lane 2), MMP-2 (lane 3), and MMP-3 (lane 4) for 90 min at 37°C. Following incubation, the supernatants were separated from the beads, subjected to SDS/12% PAGE, transferred to Hybond-C membranes, and blotted with anti-v5 antibody. (D) MMP-2/CD44 fusion protein expression promotes TGF-β activation in TA3sCD44 cells. TMLC luciferase assays were performed with serum-free coculture media from TA3sCD44 cells transiently transfected with the indicated cDNAs.

Article Snippet: Anti-MMP-9 antibody was from Oncogene (Cambridge, MA and Santa Cruz, CA) and anti-TGF-β1, TGF-β2, and TGF-β3 as well as pan-specific anti-TGF-β antibodies were from R & D Systems (Minneapolis, MN).

Techniques: Purification, Luciferase, Activity Assay, Incubation, Western Blot, Affinity Purification, Generated, Expressing, Activation Assay, Transfection

Journal: bioRxiv

Article Title: Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

doi: 10.1101/2021.11.04.467310

Figure Lengend Snippet: ( A ) Schematic drawing of a microfluidic chamber seeded with primary cortical neurons in the somatic department, transduced with AAV.EGFP virus. These neurons extend their axons through the microgrooves (length: 450 μm) into the axonal compartment within 11 days (green cells). ( B and C ) STED-images of cortical neurons stained for spectrin and tubulin. Note that spectrin is enriched in the growth cone while tubulin concentration is decreased. Scale bar 5 μm. ( D ) Quantification of line intensity scans along the axon, starting from the growth cone tip. Spectrin fluorescence intensity peaks at the growth cone, whereas the tubulin signal is lower compared to the axon. Data is represented as mean ± SD. At least 5 biological replicates were analyzed, 11 axons were analyzed in total. ( E ) Representative STED image of a growth cone stained for spectrin and F-actin. Spectrin is enriched close to the actin filaments. Scale bar 5 μm. ( F ) Schematic drawing of a growth cone, showing an enrichment of β-spectrin II in the apical central zone of the GC and microtubules splaying into the GC. ( G ) Representative STED image of a growth cone stained for F-actin and spectrin. Spectrin is enriched in the growth cone, in particular close to the base of filopodia (open arrowhead). G4 shows an enlarged version of G3 ; closed arrowheads point to enriched spectrin at the base of filopodia. Scale bar 4 μm in G1-3 and 1 μm in G4 .

Article Snippet: Monoclonal primary antibodies directed against β-spectrin II were obtained from BD (BD Bioscience, Franklin Lakes, NJ, USA; mouse, #612563, 1:200), and rabbit β-III-tubulin antibodies were from Cell Signaling (Cambridge, UK; #5568S, 1:100).

Techniques: Transduction, Virus, Staining, Concentration Assay, Fluorescence

Journal: bioRxiv

Article Title: Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

doi: 10.1101/2021.11.04.467310

Figure Lengend Snippet: Quantification of 50 μm long line intensity scans along the axon, starting from the growth cone base. Due to the mean growth cone length, the first 7 μm of the line intensities represent the GCs, whereas the other 7 – 50 μm represent the axons. β-Spectrin II-fluorescence intensity peaks at the growth cone, whereas the β-III-tubulin signal is lower compared to the axon. Data is represented as mean ± SD. At least 4 biological replicates were analyzed, 11 axons were analyzed in total.

Article Snippet: Monoclonal primary antibodies directed against β-spectrin II were obtained from BD (BD Bioscience, Franklin Lakes, NJ, USA; mouse, #612563, 1:200), and rabbit β-III-tubulin antibodies were from Cell Signaling (Cambridge, UK; #5568S, 1:100).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

doi: 10.1101/2021.11.04.467310

Figure Lengend Snippet: Cells were cultured for 11 days. On DIV 11, calpeptin or DMSO was administered into the axonal compartment for 1 hour and axotomized afterward. Regenerating axons were fixed 120 minutes later and stained for spectrin and tubulin. Axonal diameter was then measured at 10 μm from the GC. Data is given as mean ± SD. At least 7 Axons were analyzed per condition, n=3, 15 axons were analyzed in total. No significant difference was detected according to unpaired t test.

Article Snippet: Monoclonal primary antibodies directed against β-spectrin II were obtained from BD (BD Bioscience, Franklin Lakes, NJ, USA; mouse, #612563, 1:200), and rabbit β-III-tubulin antibodies were from Cell Signaling (Cambridge, UK; #5568S, 1:100).

Techniques: Cell Culture, Staining

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Effect of inhibitors of TGF β 3 , RhoA, and MEK pathways on palatal EMT. Hematoxylin and eosin-stained sections of palates fixed at different stages of palatogenesis (A–D) and after treatment with TGFβ 3 -blocking antibody (E and F), C3 (G–I) and U0126 (J–L). (A–D) Normal palatal shelves adhere to form the MEE seam by 12 h (A, arrow) Then, at 24 h the seam starts to break into small islands of epithelial cells (B). By 48 h, a few remnants of MEE are left (C). EMT is complete by 60 h (D, arrow). (E and F) Palates treated with anti-TGFβ 3 antibody (2 ng/μL) form a seam (E, arrow) by 12 h, but do not transform the MEE by 60 h (F, arrow). (G–I) Palates treated with C3, a RhoA inhibitor, form an MEE by 12 h (G, arrow) that transforms into mesenchyme by 24–48 h (H and I). The palates reach complete confluence by 60 h (not depicted). (J–L) Palates treated with U0126, a MEK1/2 inhibitor, form MEE seams that also undergo transformation (J, 24 h; K, 36 h) to complete EMT at 60 h (L, arrow). There were no effects of RhoA inhibitor (C3) or MEK inhibitor (U0126) on normal palatogenesis.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Staining, Blocking Assay, Transformation Assay

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Quantification of disappearance of cells from epithelial seam during EMT. Using LCM on 8-μm-thick frozen sections from the middle one third of the palate, we estimated cell numbers in each section of the MEE seam from 12 to 60 h after the beginning of the culture. On the ordinate, we estimated seam transformation by the number of cells remaining in it. Stage 1, 0 cells (confluence); stage 2, 1–19 cells; stage 3, 20–39 cells; stage 4, 40–59 cells; stage 5, 60–76 cells; stage 6, untransformed seam (75 cells). Palates were treated from the beginning of culture with the following reagents: TGFβ 3 -blocking antibody, TGFβ 3 -blocking antibody plus LEF1, RhoA inhibitor C3, MEK1/2 inhibitor U0126, DN LEF1 virus, DN Smad4 adenovirus, and AS β-catenin. A (a) is the untreated control. Length of time is indicated by the decoration inside the graph bars. Untreated/control palates are confluent by 60 h (A, a), and palates treated with anti-TGFβ 3 antibody do not transform (A, b). Treatment with C3 (A, c), U0126 (A, d), and AS β-catenin (B, d) have no effect on MEE seam transformation. LEF1 does not rescue blockage by anti-TGFβ 3 antibody (B, a). Treatment with DN LEF1 (B, b) or DN SMAD4 (B, c) inhibits transformation.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Transformation Assay, Blocking Assay, Virus

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: LEF1 gene expression (real-time quantitative PCR). LEF1 mRNA expression was measured by highly sensitive real-time PCR in LCM-dissected MEE cells from frozen sections. (A) Palates were treated with exogenous LEF1 virus, anti-TGFβ 3 antibody, DN LEF1 virus, and exogenous recombinant TGFβ 3 . (B) Palates were treated with exogenous LEF1 virus plus anti-TGFβ 3 -blocking antibody, AS β-catenin oligodeoxynucleotide, DN Smad4 adenovirus, C3, a Rho inhibitor, and U0126, a MEK1/2 inhibitor. Length of time is indicated within each graph bar, as in . (A, a) In the untreated palates (Untr/Cntrl), LEF1 expression peaks at 36 h, at which time EMT also peaks. (A, b) In presence of exogenous LEF1, there is a large increase in LEF1 mRNA expression that peaks at 24 h instead of 36 h. (A, c) TGFβ 3 inhibition by blocking antibody causes complete inhibition of LEF1 mRNA expression and EMT (A, d) DN LEF1 causes palates to cease endogenous LEF1 mRNA expression and remain untransformed. (A, e) Exogenous TGFβ 3 causes LEF1 mRNA and protein to up-regulate and reach a peak at 24 h without significantly hastening the EMT process. (B, a) Exogenous LEF1 DNA increases LEF1 mRNA, but the seam remains untransformed if palates are treated with TGFβ 3 -blocking antibody. (B, b) AS β-catenin did not affect LEF1 mRNA expression as compared with the control (A, a). (B, c) DN Smad4 inhibits LEF1 mRNA expression and MEE remains untransformed. (B, d) RhoA inhibitor C3 had no effect on normal LEF1 mRNA expression or EMT, (B, c) and neither did MEK1/2 inhibitor U0126 (B, e).

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Virus, Recombinant, Blocking Assay, Inhibition

Journal: The Journal of Cell Biology

Article Title: TGFβ 3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development

doi: 10.1083/jcb.200306024

Figure Lengend Snippet: Immunolocalization of Smad2, phospho-Smad2, and Smad4 in untreated and treated (TGF β 3 -blocking antibody and DN Smad4) palates. (A) Phospho-Smad2 is intensely present in the nuclei of normal palatal MEE (12 h), indicating it is translocated by Smad4 into the nucleus (arrow) during normal palatogenesis. (B) Phospho-Smad2 in MEE of palates treated (36 h) with DN Smad4 is located in the cytoplasm and no nuclear localization occurs (arrow), confirming that Smad4 is required for nuclear translocation of phospho-Smad2. (C) Unphosphorylated Smad2 is located in the cytoplasm of MEE cells not in the nucleus (arrow) of palates lacking Smad4 after 12 h of culture. (D) Smad2 is also located in cytoplasm of the MEE cells (36 h), but not in the nucleus (arrow) in the presence of blocking TGFβ 3 , confirming that TGFβ 3 is required for the phosphorylation of Smad2. (E) Smad2 in MEE of palates (36 h) treated with DN Smad4 is located in the cytoplasm of the cell, but not in the nucleus (arrow). At this late phase, the MEE is still untransformed. (F) DN Smad4 inhibits the transport of Smad4 itself to the nucleus. Thus, nuclear Smad2/4 is not available to activate LEF1, and these palates do not transform.

Article Snippet: The linearized recombinant plasmid was packaged into infectious adenoviral particles by transfecting HEK 293 cells using LipofectAMINE™ PLUS reagent, and the recombinant adenovirus was harvested after 7–10 d. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy. (4) 2 ng/μl anti–mouse TGFβ 3 -blocking antibody (R&D Systems). (5) 2 ng/μl recombinant TGFβ 3 (R&D Systems). (6) AS β-catenin oligonucleotides 5′-GGAGTTTAACCACAACAGGCAGTCC-3′ and control sense 5′-CCTGACGGACAACACCAATTTGAGG-3′, as described by , used by , were tested in our laboratory. (7) 15 μM MEK1/2 inhibitor UO126 (Cell Signaling). (8) RhoA inhibitor C3 (Calbiochem).

Techniques: Blocking Assay, Translocation Assay

Journal:

Article Title: Targeted disruption of TGF-?1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction

doi: 10.1172/JCI200319270

Figure Lengend Snippet: Lack of Smad3 prevents renal fibrosis, monocyte influx, and TGF-β1 upregulation. (a and b) Immunofluorescence of type I collagen in obstructed kidneys of WT (a) and Smad3-null (KO) (b) mice at day 14 after UUO. (c) Hydroxyproline content in obstructed kidneys from WT and KO mice and sham-operated WT mice (Sham). (d and e) Immunofluorescence of F4/80 antigen, a mouse monocyte marker, in obstructed kidneys from WT (d) and KO (e) mice at day 14 after UUO. DAPI (blue) was used for nuclear staining. Scale bars: 20 μm. (f) Number of monocytes per unit area in obstructed kidneys from WT and KO mice with UUO and sham-operated WT mice (Sham). (g) Northern blot of TGF-β1 mRNA in kidneys from WT and KO mice with UUO and sham-operated counterparts (Sham). (h) Active and total TGF-β1 concentrations as determined by immunoassay in kidneys of WT and KO mice and sham-operated mice (Sham). Results are means ± standard deviation of four to five samples. *P < 0.01 compared with Sham or KO.

Article Snippet: Mouse monoclonal anti–TGF-β1, -β2, -β3 antibody (clone 1D11; R&D Systems) was used to neutralize TFG-β bioactivity at a concentration of 20 μg/ml, with mouse IgG (Sigma Aldrich, St. Louis, Missouri, USA) as a control.

Techniques: Immunofluorescence, Marker, Staining, Northern Blot, Standard Deviation

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A . Immunoblot analysis of the short form of the Sirt3 was performed in sham and AngII-treated WT and Sirt3-KO mice hearts. Tubulin expression was used as loading control. B . Ratio of the heart weight to body weight in WT and Sirt3-KO mice infused with either saline or AngII for 4 weeks. (n=5) C . The left ventricular wall thickness was measured with echocardiology as described in the methods section. (n=5) D-E . Hematoxylin/eosin stained cardiac sections from control or AngII-treated WT and Sirt3-KO mice showed cardiomyocyte loss or dropout. Masson's trichrome stained sections of the hearts were to detect fibrosis (blue). The graph showed the quantification of interstitial fibrosis in sham or AngII-treated WT and Sirt3-KO mice. (n=5) Scale bar: 20 μm. F . ANF and Myh7 mRNA levels in heart samples of sham or AngII-treated WT and Sirt3-KO mice. (n=5) The data are presented as the means ± SEM of three independent experiments. *P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Saline, Staining

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A . Immunoblot analysis of Sirt3, LC3 was performed on primary neonatal rat cardioyocytes treated with AngII (1μM, 24h) or chloroquine (CQ, 60μM, 16h). Tubulin expression was used as loading control. B-C . Immunoblot analysis of Sirt3 and autophagic markers was performed on primary neonatal cardioyocytes from WT and Sirt3-KO mice. Tubulin expression was used as loading control. Bar graphs showed the quantification of LC3-II, Beclin-1 and p62 measured by densitometry analysis. (n=5) D-E . The H9C2 cardiomyocytes were transfected with siRNA-Sirt3, and then treated with CQ and AngII. GAPDH expression was used as loading control. Bar graph represents quantification of LC3-II levels measured by densitometry analysis. (n=5) F . The bar graph showing the quantification of ANF and Myh7 mRNA levels as in D. (n=5) The data are presented as the means ± SEM of three independent experiments.*P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Transfection

Journal: Oncotarget

Article Title: Mouse Sirt3 promotes autophagy in AngII-induced myocardial hypertrophy through the deacetylation of FoxO1

doi: 10.18632/oncotarget.13429

Figure Lengend Snippet: A-B . Immunoblot analysis of FoxO1 and ac-FoxO1 was performed in sham and AngII-treated WT and Sirt3-KO murine hearts. Tubulin expression was used as loading control. Bar graph represents quantification of ac-FoxO1 levels measured by densitometry analysis. (n=5) C-D . Immunoblot analysis of autophagy biomarkers and Sirt3 in the control and siRNA-FoxO1 groups. GAPDH expression was used as loading control. Bar graph represents quantification of LC3-II levels measured by densitometry analysis. (n=5) E . Cellular extracts were immunoprecipitated with Sirt3 antibody and analyzed with anti-FoxO1. F . Cellular extracts were immunoprecipitated with FoxO1 antibody and analyzed with anti-Sirt3. The data are presented as the means ± SEM of three independent experiments.*P<0.05, **P<0.01.

Article Snippet: Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.

Techniques: Western Blot, Expressing, Control, Immunoprecipitation