Journal: Molecular Cancer

Article Title: New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

doi: 10.1186/1476-4598-9-192

Figure Lengend Snippet: Cytotoxic effects of the SB-T-1214 against colonospheres grown in 3D cultures . Ten day-old colonospheres grown from FACS sorted CD133 high /CD44 high HCT116 cells on the ultra-low attachment 6-well plates (Corning) were treated with 100 nM SB-T-1214 for 48 hours, then drug-containing medium was replaced with regular MSCBM and analyzed after 24 hours. Treatment with drug induced a profound loss of sphere integrity (A, C) and apoptosis in the majority of the sphere cells ( D-E; FACS analysis with the annexin V-FITC and propidium iodide). The FITC-conjugated drug showed efficient penetration into spheroid after 30 min of incubation (B). To evaluate possible alterations in sphere-forming capacity, control and drug treated spheroids/cells were dissociated to a single cell suspension and an equal number of viable cells (400 cells) were plated on ULA 6-well plates in MSCB medium containing 10% Matrigel. The number of secondary spheroids was counted ten days after plating (F).

Article Snippet: Four hunderd cells of particular phenotype were seeded on each well of the Ultra-Low Attachment (ULA) 6-well plate (Corning, Lowell, MA) in serum-free MSCB medium containing 10-25% Matrigel matrix (BD Biosciences) and examined after 1 week of culturing under standard conditions.

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: Low doses of SB203580 have a negligible effect on the proliferation, viability and apoptosis of HBFs AS. HBF populations ( n = 3) were exposed to increasing concentrations (5–20 μM) of SB203580 for one, three and five days, each in triplicates. ( A ) The proliferation rate of HBFs measured by crystal violet assay. ( B ) The viability of HBFs determined with the MTT test. Results were presented as a percent of control cells cultured in serum-free medium without SB203580 exposition by one, three or five days. The percentage of apoptotic cells in HBF populations (30,000 events) treated by SB203580 (10 µM) for five days determined with Annexin V/PI assay and flow cytometry presented on ( C ) the dot plot (Q1: Early-apoptotic cells—green, Q2; Late-apoptotic cells—red, Q3: Live cells-blue, Q4: Necrotic cells—red) and ( D ) the graph showing the mean ± SEM. Statistical significance was tested using the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test; * p < 0.05; ** p < 0.01.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Crystal Violet Assay, Control, Cell Culture, Flow Cytometry, Comparison

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: The TGF-β 1 -induced FMT of HBFs AS is significantly inhibited by SB203580. The efficiency of the FMT was checked in HBF AS populations cultured without or with SB203580 (10 μM) in the absence or presence of TGF-β 1 (5 ng/mL) for five days. ( A ) Representative images of HBFs AS, immunostained for α-SMA (green) and counterstained for DNA (blue), were obtained. Scale bar = 50 µm. ( B ) The percentage of myofibroblasts in HBF AS cultures ( n = 11) are presented on the graph. The level of α-SMA in HBFs AS ( n = 3) was determined using ( C , D ) Western blot and ( E ) in-cell ELISA. RCU—relative colorimetric units. ( F , G ) Relative expression levels of myofibroblast-related genes: ACTA2 (encoding α-SMA), TAGLN (encoding transgelin) in HBFs AS ( n = 4) cultured for 24 h in conditions described above were presented as a 2 −ΔΔCt mean value in relation to control genes ( 18S , B2M and GAPDH ) and control conditions. Statistical significance was tested using the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Cell Culture, Western Blot, In-Cell ELISA, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: ECM-related myofibroblast markers were significantly reduced by SB203580 in TGF-β 1 -treated HBF AS cultures. HBF AS populations were cultured without or with SB203580 (10 μM) in the absence or presence of TGF-β 1 (5 ng/mL) for five days. ( A ) Representative images of HBFs AS, immunostained for collagen1 (magenta) and counterstained for DNA (blue), were obtained. Scale bar = 50 µm. The levels of collagen1 in HBFs AS cultures ( n = 3) were determined ( B ) fluorometrically, ( C ) using in-cell ELISA and ( D , E ) Western blot. The levels of fibronectin in the HBF AS cultures ( n = 3) were determined ( G ) using in-cell ELISA and ( D , H ) Western blot (representative membranes are shown). Relative expression levels of ( F ) COL1A1 (encoding collagen 1A1), COL1A2 (encoding collagen 1A2) and ( I ) FN1 (encoding fibronectin) in the HBFs AS ( n = 4) cultured for one day in conditions described above were presented as a 2 −ΔΔCt mean value in relation to control genes ( 18S , B2M and GAPDH ) and control conditions. RCU—relative colorimetric units. Statistical significance was tested using the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Cell Culture, In-Cell ELISA, Western Blot, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: SB203580 affected the vinculin-rich and integrin-mediated adhesion sites in TGF-β 1 -treated HBFs AS. HBF AS populations ( n = 3) were cultured without or with SB203580 (10 μM) in the absence or presence of TGF-β 1 (5 ng/mL) for five days. Then, the HBFs AS were immunostained for vinculin (green), counterstained for F-actin (red) and DNA (blue), and the vinculin-rich FAs were imaged using a fluorescence microscope with a TIRF module. ( A ) Representative images of FAs were presented (scale bar = 25 µm), the marked box has been enlarged and presented in the bottom row, ( B ) quantified (average (AVG) length of at least 200 FAs per condition in boxes ± SEM) and grouped by size. ( C ) The protein levels of vinculin and integrin αV in HBF AS cultures ( n = 3) were determined by Western blot and ( D , E ) quantified. Representative membranes were shown. ( F ) Relative expression levels of ITGA5 (encoding integrin α5) in HBFs AS ( n = 4) cultured for one day in conditions described above were presented as a 2 −ΔΔCt mean value in relation to control genes ( 18S , B2M and GAPDH ) and control conditions. Statistical significance was tested using the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test; * p < 0.05; *** p < 0.001; ns—not statistically significant.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Cell Culture, Fluorescence, Microscopy, Western Blot, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: SB203580 significantly reduced the level of Cx43 in TGF-β 1 -treated HBFs AS. HBF AS populations were cultured without or with SB203580 (10 μM) in the absence or presence of TGF-β 1 (5 ng/mL) for five days. ( A ) Representative images of HBFs AS, immunostained for Cx43 (red) and counterstained for DNA (blue), were obtained. Scale bar = 50 µm. The levels of Cx43 in HBF AS cultures were determined ( B ) fluorometrically ( n = 11), ( C ) using in-cell ELISA ( n = 4) and ( D ) Western blot with ( E ) densitometric analysis ( n = 3). A representative membrane was shown. ( F ) The relative expression of GJA1 (encoding Cx43) in HBFs AS ( n = 4) cultured for one day in conditions described above was presented as a 2 −ΔΔCt mean value in relation to control genes ( 18S , B2M and GAPDH ) and control conditions. RCU—relative colorimetric units. Statistical significance was tested using the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Cell Culture, In-Cell ELISA, Western Blot, Membrane, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

doi: 10.3390/ijms222312790

Figure Lengend Snippet: SB203580 affected the TGF-β 1 -induced phosphorylation levels of Smad2 and Smad3 in the cell nuclei area and diminishes the phosphorylation of p38 MAPK in HBFs AS. HBF AS populations were cultured without or with SB203580 (10 μM) in the absence or presence of TGF-β 1 (5 ng/mL) for one hour. ( A ) Representative images of HBFs AS, immunostained for pSmad2 and pSmad3 (red) and DNA (blue), were presented. Scale bar = 50 µm. ( B ) The mean fluorescence intensity (±SEM) of pSmad2 and ( C ) pSmad3 in the cell nuclei area was measured using ImageJ ( n = 3; at least 100 cells per condition). ( D ) The levels of p-p38 and p38 MAPK in HBF AS cultures ( n = 3) were determined using Western blot and quantified in ( E ). Representative membranes were shown. The statistical significance of the differences between conditions was determined by the non-parametric Kruskal–Wallis test with Dunn’s Multiple Comparison post hoc test, * p < 0.05; ** p < 0.01; *** p < 0.001; ns—not statistically significant.

Article Snippet: The HBFs (1 × 10 4 per cm 2 ) were cultured in 6-well plates for 24 h in a complete medium, which was changed for a serum-free medium without or with SB203580 (10 μM) alone or in combination with TGF-β 1 (5 ng/mL; Corning, NY, USA).

Techniques: Phospho-proteomics, Cell Culture, Fluorescence, Western Blot, Comparison