Journal: Pharmaceutics

Article Title: Improving the Culture of Human Skin Explants for Use in Preclinical Testing of Wound Healing Treatments

doi: 10.3390/pharmaceutics17121611

Figure Lengend Snippet: A schematic representation of the standardized ex vivo wound model and of the experimental design. Skin samples were cut into 1.5 × 1.5 cm pieces, and superficial wounds with a diameter of 3 mm were created. The wounded skin samples were transferred into inserts and cultured in four different media with different supplementation: (i) DMEM with FCS, (ii) DMEM with NHS and OC, (iii) CnT medium with NHS and OC, and (iv) EpiLife™ medium with NHS and OC. A total of five plates containing skin samples were cultured at 37 °C, 5% CO 2 for 1, 3, 6, 9, and 12 days. At 3-day intervals, one skin sample and 1 mL of culture medium per group were collected. One half of the skin samples was used for histology (H&E staining), immunofluorescence staining (Keratin 17 (K17) and CD31), and immunohistochemical (IHC) staining (type I and type III collagen, TGFβ2, and MMP-9). The other half was processed for protein extraction and ELISA analysis to quantify K17 and CD31 expression, as well as for caspase-3/7 activity assays. The collected culture media were analyzed for lactate dehydrogenase (LDH) activity and measurement of IL-1α, IL-6, and IL-8 by ELISA.

Article Snippet: The concentrations of proinflammatory cytokines IL-1α, IL-6, and IL-8 in the culture media were measured using commercially available ELISA kits (IL-1α: R&D Systems, #DY200; IL-6 and IL-8: CytoSetTM CHC1263 and CHC1303, Invitrogen Corporation, Carlsbad, CA, USA), following the manufacturers’ instructions.

Techniques: Ex Vivo, Cell Culture, Staining, Immunofluorescence, Immunohistochemical staining, Immunohistochemistry, Protein Extraction, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay

Journal: Cancers

Article Title: Inhibition of FGF2-Mediated Signaling in GIST—Promising Approach for Overcoming Resistance to Imatinib

doi: 10.3390/cancers12061674

Figure Lengend Snippet: KIT inhibition induces the changes in gastrointestinal stromal tumor (GIST) secretome. ( A ) Median chemokine expression levels in the cell culture supernatants of imatinib mesylate (IM)-treated (1 µmol/L) GIST T-1 cells compared with non-treated controls. ( B ) Median chemokine expression levels in the cell culture supernatants of IM-resistant GIST T-1R cells treated with IM (1 µmol/L) compared with non-treated controls. ( C ) FGF-2 levels in supernatants of IM-naive vs. resistant GISTs treated with IM (1 µmol/L) for 2–6 days. ( D ) Comparative assessment of FGF-2 levels in cell culture supernatants of GIST T-1R cells treated with IM (1 µmol/L) or transfected with siRNA KIT . ( E ) Comparative assessment of IL-6, MCP-3, and GRO levels in cell culture supernatants of GIST T-1R cells treated with IM (1 µmol/L) or transfected with siRNA KIT . ( F ) Changes in the relative expression level of FGF2 in GIST T-1 vs. T-1R cells treated with IM (1 µmol/L) alone or in the presence of BGJ398 (1 µmol/L), as determined by quantitative RT-PCR. For internal control, the amplification of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was used. ( G ) Comparative assessment of FGF-2 levels in cell culture supernatants of GIST T-1R cells treated with IM (1 µmol/L) alone or in the presence of BGJ398 (1 µmol/L). Data are presented as median ± SD. Significant differences with p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) from n ≥ 3 using unpaired Student’s t -test.

Article Snippet: The levels of FGF2 in the serum of mice bearing IM-naive and -resistant GIST xenografts were measured using a human FGF2 ELISA Kit (R&D Systems, Tustin, CA, USA), according to the manufacturer’s protocol.

Techniques: Inhibition, Expressing, Cell Culture, Transfection, Quantitative RT-PCR, Control, Amplification

Journal: Cancers

Article Title: Inhibition of FGF2-Mediated Signaling in GIST—Promising Approach for Overcoming Resistance to Imatinib

doi: 10.3390/cancers12061674

Figure Lengend Snippet: IM stimulates invasion, migration, and colony formation in GIST T-1R cells. ( A ) Matrigel transwell invasion assay representative images of GIST T-1R cells treated with vehicle, IM (1 µmol/L) alone or in the presence of BGJ398 (1 µmol/L), a selective FGFR inhibitor, or anti-FGF-2 neutralizing Abs (20 µg/mL). ( B ) Matrigel transwell invasion assay quantification as the number of invading GIST T-1R cells per microscopic field after treatment with vehicle, IM (1 µmol/L) alone or IM in the presence of BGJ398 (1 µmol/L) or anti-FGF-2 Abs (20 µg/mL). ( C ) Representative images of the wound healing assay of GIST T-1R cells upon IM treatment (1 µmol/L) for 48 h alone or in the presence of BGJ398, a selective FGFR-inhibitor (1 µmol/L) or anti-FGF2 neutralizing Abs (20 µg/mL). GIST cells treated with vehicle were used as control. ( D ) Quantitative analysis of wound area GIST T-1R cells treated with DMSO (control), IM alone or in presence of FGF-2 or BGJ398; ( E ) Representative images of the colony formation assay of GIST T-1R cells upon IM treatment (1 µmol/L) for 48 h alone or in the presence of BGJ398, a selective FGFR-inhibitor (1 µmol/L) or anti-FGF-2 neutralizing Abs (20 µg/mL). GIST cells treated with vehicle were used as a control. ( F ) Quantification of the colonies in GIST T-1R cells treated with DMSO (control), IM alone or in presence of FGF-2 or BGJ398. Data are presented as median ± SD. Significant differences with p < 0.05 (*), p < 0.01 (**) from n ≥ 3 using unpaired Student’s t -test.

Article Snippet: The levels of FGF2 in the serum of mice bearing IM-naive and -resistant GIST xenografts were measured using a human FGF2 ELISA Kit (R&D Systems, Tustin, CA, USA), according to the manufacturer’s protocol.

Techniques: Migration, Transwell Invasion Assay, Wound Healing Assay, Control, Colony Assay

Journal: Cancers

Article Title: Inhibition of FGF2-Mediated Signaling in GIST—Promising Approach for Overcoming Resistance to Imatinib

doi: 10.3390/cancers12061674

Figure Lengend Snippet: Inhibition of FGF-signaling deregulates AKT- and MAPK-phosphorylation in IM-resistant GISTs. ( A ) Anti-FGF2 neutralizing Abs (20 µg/mL) and BGJ398 (1 µmol/L) abrogate IM-induced activation of GIST T-1R cells. Cells were treated with IM (1 µmol/L) in the presence of anti-FGF-2 Abs for 72 h and subjected to the immunoblot analysis for total and phosphorylated forms of FGFR, MAPK, and AKT. Actin staining was used to show the comparable amounts of protein loaded into each sample. ( B ) Quantification by mean pixel density revealed that the phosphorylated proteins are dysregulated in GIST T-1R after the inhibition of FGFR signaling. ( C ) The impact of anti-FGF2 neutralizing Abs (20 µg/mL) and BGJ398 on IM-induced activation of GIST 430 cells. Cells were treated with IM (1 µmol/L) in the presence of anti-FGF-2 Abs for 72 h and subjected to immunoblot analysis for total and phosphorylated forms of FGFR, MAPK, and AKT. Actin staining was used to show the comparable amounts of protein loaded into each sample. ( D ) Quantification by mean pixel density revealed that the phosphorylated proteins are dysregulated in GIST 430 after the inhibition of FGFR signaling. The whole western blot of A,C are in .

Article Snippet: The levels of FGF2 in the serum of mice bearing IM-naive and -resistant GIST xenografts were measured using a human FGF2 ELISA Kit (R&D Systems, Tustin, CA, USA), according to the manufacturer’s protocol.

Techniques: Inhibition, Phospho-proteomics, Activation Assay, Western Blot, Staining

Journal: Bone Reports

Article Title: Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants

doi: 10.1016/j.bonr.2020.100715

Figure Lengend Snippet: Markers of cartilage synthesis and turnover in patients with primary total hip arthroplasty (THA) vs healthy (A) Type II procollagen, ** p = 0.004 THA 6 W vs healthy 58Y, §§ p = 0.002 THA 6 W vs THA PR (B) PIICP, ** p = 0.003 THA 3 M vs healthy 58Y, §§ p = 0.005 THA 3 M vs THA PR (C) CTX-II, * p = 0.011 THA 6 W vs healthy 58Y, *** p < 0.001 THA 5Y vs healthy 67Y, §§ p = 0.002 THA 6 W vs THA PR, §§§ p < 0.001 THA 5Y vs THA PR. Mean ± SEM.

Article Snippet: Type II procollagen: CA/DA/hRP (DuoSet, Biotechne R&D Systems, cat.no.DY7589).

Techniques:

Journal: Bone Reports

Article Title: Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants

doi: 10.1016/j.bonr.2020.100715

Figure Lengend Snippet: MMPs, ADAMTS, serine proteases and endogenous inhibitors in the three groups of healthy. Mean ± SEM.

Article Snippet: Type II procollagen: CA/DA/hRP (DuoSet, Biotechne R&D Systems, cat.no.DY7589).

Techniques: