Journal: Cell Death and Differentiation

Article Title: Autologous micrograft accelerates endogenous wound healing response through ERK-induced cell migration

doi: 10.1038/s41418-019-0433-3

Figure Lengend Snippet: The in vivo healing potential of AMG through ERK signaling pathway. a Schematic representation of the in vivo WH study performed on C57BL/6 mice. Animals were topically treated with vehicle, AMG, and MEK inhibitor—trametinib (0.2 mg) on day 0, 2, 4, and 6. At the end of the experiment (day 8), skin wound samples were collected for further analyses. b Excisional wound-splinting assay showing the potential of AMG to improve wound closure in AMG-treated mice compared with the other conditions under study (vehicle, AMG + trametinib and trametinib). Moreover, two out of five AMG-treated animals showed hair surrounding the wounds. c Percentage of wound closure between the groups under study. d Representative hematoxylin and eosin (H&E) stained section on day 8 after wounding. Scale bar 500 μm. Arrowheads delimitate the wound area and 2x zoomed region of interest is reported on the right. Pink dotted lines and arrowheads delimitate the epithelial tongues. e , f Percentage (%) of re-epithelialization and granulation tissue formation expressed in arbitrary units (AU) among the evaluated groups. Re-epithelialization coverage in wounds treated with AMG reached 83% ± 10% compared with only 56% ± 4% in vehicle-treated mice. Percentage of GT formation of AMG-treated group was significantly higher (70% ± 2,7%) compared with the other conditions, suggesting increased fibroplasia in AMG-treated mice. g Representative Sirius Red (SR) stained section on day 8 after wounding. Scale bar 100 μm. h , i % of total collagen formation and % organized collagen on the total collagen was evaluated between the conditions. j Representative immunofluorescence of vehicle and AMG-treated wounds exposed or not to trametinib, showing presence of CD31 + and ɑ-SMA + vessels. Scale bar 200 μm. Arrowheads delimitate the wound area and 2x zoomed region of interest is reported on the right. Arrows indicate CD31 + vessels. k Number of CD31 + per mm 2 tissue and ɑ-SMA coated CD31 + vessels were evaluated in the entire wound area. l Representative immunofluorescence of vehicle and AMG-treated wounds showing the presence of the proliferative marker Ki67. % of Ki67 + cells were evaluated in both dermal (White region—I) and epidermal (Red region—II) layers ( N = 4). Scale bare 500 μm. Arrowheads delimitate the wound area and 2x zoomed region of interest is reported above the images. m No significant differences were found in the percentage of Ki67 + cells in the dermal layer. Two-tailed unpaired t test was used to perform statistical analysis. n Percentage of Ki67 + cells in the epidermal layer. Two-tailed unpaired t test was used to perform statistical analysis ( p < 0,08). o MMP gene expression evaluated by RT-qPCR in vehicle and AMG-treated in presence or absence of trametinib. Expression values are expressed as a z-score of the average fold change (FC) normalized on the expression of Gapdh , B-actin, and Rpl13a housekeeping genes ( N = 3). p Representative immunohistochemistry for pERK in vehicle and AMG-treated in presence or absence of trametinib. Scale bar 100 μm. Quantification of pERK + cells (%) analyzed using QuPath software. All data are presented as mean ± SEM. Differences are calculated using one-way ANOVA ( N = 5) and indicated as * P < 0.05; *** P < 0.005; **** P < 0.001

Article Snippet: A second group was topically treated with AMG supplemented with the specific MEK inhibitor—trametinib (0.2 mg, Cat No. HY-10999, MCE).

Techniques: In Vivo, Staining, Immunofluorescence, Marker, Two Tailed Test, Gene Expression, Quantitative RT-PCR, Expressing, Immunohistochemistry, Software

Journal: International Journal of Oncology

Article Title: Survivin knockdown induces senescence in TTF-1-expressing, KRAS -mutant lung adenocarcinomas

doi: 10.3892/ijo.2018.4365

Figure Lengend Snippet: Combination of survivin knockdown with ABT-263 and trametinib effectively induces apoptosis of KRAS -mutant lung adenocarcinoma cells. (A) Effects of triple combination therapy of survivin knockdown, ABT-263 and trametinib on the viability of H358 and H441 cells. Cells were transfected with BIRC5 siRNA #1 or #2 (10 nM each) and cultured for 48 h. Cells were then treated with the ABT-263 (1 μ M) alone, trametinib (25 nM) alone, both, or neither for a further 72 h. Results are shown as the means ± SD; ** P<0.01. (B) Crystal violet staining of viable cells. NC siRNA or BIRC5 siRNA #1 (10 nM each)-transfected cells were grown for 48 h. A total of 5×10 5 cells were then seeded in 6-well plates and treated with ABT-263 (1 μ M) alone, trametinib (25 nM) alone, both, or neither for a further 72 h. Cells were then fixed and stained with crystal violet. (C) The effects of triple combination therapy of survivin knockdown, ABT-263 and trametinib on caspase 3/7 activity in H358 and H441 cells. The NC siRNA- or BIRC5 siRNA #1 (10 nM each)-transfected cells were cultured for 48 h, and the cells were then treated with ABT-263 (1 μ M) alone, trametinib (25 nM) alone, both, or neither for a further 24 h before evaluating caspase activity. Caspase 3/7 activity was normalized to 100 for the mean of three control (NCsi) dishes. Columns, mean (n=3); bars, SD; ** P<0.01. (D) Western blot analysis of the effects of the triple combination therapy on H358 and H441 cells. Cells were treated in the same manner as described in (A) or (B). Of note, Bim (EL) was dephosphorylated by trametinib treatment, and then accumulated in the cell. EL, extra-long; L, long; S, short.

Article Snippet: The MEK inhibitor, trametinib (AdooQ BioScience, Irvine, CA, USA) and the Bcl-2 inhibitor (also known as a BH3 mimetic drug), ABT-263 (AdooQ BioScience), were also used in this study.

Techniques: Knockdown, Mutagenesis, Transfection, Cell Culture, Staining, Activity Assay, Control, Western Blot

Journal: International Journal of Oncology

Article Title: Survivin knockdown induces senescence in TTF-1-expressing, KRAS -mutant lung adenocarcinomas

doi: 10.3892/ijo.2018.4365

Figure Lengend Snippet: Triple combination therapy comprising of survivin knockdown, ABT-263 and trametinib is also effective against three-dimensionally (3D) cultured KRAS -mutant lung adenocarcinoma cells. (A) Schematic description of the experimental procedure and the representative image of a microspheroid in the 3D 'on-top' culture condition. (B) Representative images of microspheroids of 50 cells in 3D 'on-top' culture condition. The spheroids derived from NC siRNA- or BIRC5 siRNA (10 nM each)-transfected cells were cultured in the absence or presence of ABT-263 (1 μ M) plus trametinib (25 nM) for 96 h. (C) Effects of the triple combination therapy on H358 and H441 spheroids. Viability was assessed as described in the schema in (A). Results are shown as the means ± SD; *P<0.05, ** P<0.01.

Article Snippet: The MEK inhibitor, trametinib (AdooQ BioScience, Irvine, CA, USA) and the Bcl-2 inhibitor (also known as a BH3 mimetic drug), ABT-263 (AdooQ BioScience), were also used in this study.

Techniques: Knockdown, Cell Culture, Mutagenesis, Derivative Assay, Transfection

Journal: International Journal of Oncology

Article Title: Survivin knockdown induces senescence in TTF-1-expressing, KRAS -mutant lung adenocarcinomas

doi: 10.3892/ijo.2018.4365

Figure Lengend Snippet: Triple combination of survivin knockdown, ABT-263, and trametinib decreases the size of xenografts of H358 cells. (A) Effects of the triple combination therapy on xenografts of H358 cells. Mice with tumor xenografts derived from BIRC5 siRNA-transfected cells were untreated (n=5) or treated (n=5) with ABT-263 (50 mg/kg) and trametinib (0.6 mg/kg) for 22 days. Mice with NC siRNA-transfected xenografts (n=5) were treated with vehicle. Drugs were administered once daily by oral gavage. Tumor volumes (means ± SD) were measured after the initiation of the AT treatment (day 1). 'Day 1' in this figure corresponds to the day 10 after implantation of the cells, when the AteloGene was also administered. Of note, one of the five mice with BIRC5 siRNA-transfected xenografts receiving AT treatment died during the experiment. The death at day 13 was probably due to the side-effects of trametinib used, as only this mouse developed a severe skin rash from day 10, when the administration to the mouse was terminated. As previously shown in clinical practice, the use of trametinib can lead to the development of skin rash . * P<0.05, ** P<0.01. (B) Effects of the triple combination therapy on xenografts derived from H358 cells. Macroscopic images of the tumors resected from mice are represented. Fourteen tumor nodules presented herein were derived from the 14 mice that survived to the end of the therapy. (C) Effect of the triple combination therapy on xenografts of H358 cells. The weight of each tumor, shown in (B), was measured, and the results are presented as the means ± SD. * P<0.05, ** P<0.01. (D) Pathological examination of xenografts. Hematoxylin and eosin (H&E) staining and immunohistochemistry for survivin of xenografts in each treatment condition. Of note, tumors derived from both the NC si-transfected and BIRC5 si-transfected cells without AT therapy expressed survivin in the nuclei, whereas the staining observed in the tumors derived from BIRC5 si-transfected cells with AT therapy were likely to reflect immunoglobulins derived from the mouse; i.e., non-specific staining.

Article Snippet: The MEK inhibitor, trametinib (AdooQ BioScience, Irvine, CA, USA) and the Bcl-2 inhibitor (also known as a BH3 mimetic drug), ABT-263 (AdooQ BioScience), were also used in this study.

Techniques: Knockdown, Derivative Assay, Transfection, Staining, Immunohistochemistry