Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: mTOR (Mechanistic Target of Rapamycin) Inhibition Decreases Mechanosignaling, Collagen Accumulation, and Stiffening of the Thoracic Aorta in Elastin-Deficient Mice

doi: 10.1161/atvbaha.117.309653

Figure Lengend Snippet: Figure 4. Temporal effects of mTOR (mechanistic target of rapamycin) inhibition on mechanosignaling. A, Bacterial artificial chromosome coding human ELN (hBAC)-null for murine Eln (mNULL) mice were treated with everolimus at 1.5 mg/kg PO every 12 h (ie, at 3 mg/kg per day) for 0 to 7 d, staggering the initial doses such that all treatments were completed at 6 wk of age. B, Alternatively, 4-wk-old hBAC- mNULL mice were treated with INK128 at 1.5 mg/kg PO every 12 h for 1, 3, or 7 doses (dual mTORC1 and mTORC2 inhibition at all time points) and compared with vehicle alone for 3 doses, everolimus at 1.5 mg/kg PO every 12 h for 3 doses (selective mTORC1 [mTOR com- plex 1] inhibitor at this treatment duration), and PF562271 at 25 mg/kg PO every 12 h for 3 doses (selective FAK inhibitor). The animals were euthanized 1 h after the last dose of drug, the thoracic aortas were rapidly procured, and extracted protein was immunoblotted for phosphorylated and total levels of S6K, S6, AKT, FAK, SMAD2, type III collagen (Col III), smooth muscle α-actin (SMA), and GAPDH; n=2 per group.

Article Snippet: Imatinib (Novartis) was used at 200 mg/kg/d [7], metformin (Sigma-Aldrich) at 300 mg/kg/d [8], INK128 (Selleck Chemicals) at 3 mg/kg/d [9], and PF562271 (Selleck Chemicals) at 50 mg/kg/d [10].

Techniques: Inhibition

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: Changes in the biological behaviors of AT2 cells. (A and C) Wound healing, (B and E) invasion assays and (D) adhesion assay. The biological behaviors of the AT2 cells were deteriorated by CS, and the deterioration was reversed following overexpression of SOX11, or aggravated by knockdown of SOX11. Furthermore, the effects of SOX11 overexpression were inhibited by a FAK antagonist. (A) Magnification, x40; (B) magnification, x100. Data are presented as the means ± standard error of the mean. * P<0.05. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Cell Adhesion Assay, Over Expression, Knockdown

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: Apoptosis of AT2 cells in each group. (A-H) Flow cytometric analysis of cell apoptosis; (I) Quantification of cell apoptosis. The graph shows the percentage of late apoptotic cells (upper right quadarant in plots). CS significantly increased the apoptosis of AT2 cells. This increase in apoptosis was attenuated by SOX11 overexpression, and aggravated by SOX11 knockdown. In addition, FAK antagonism inhibited the effects of SOX11 overexpression. Data are presented as the means ± standard error of the mean. ** P<0.01. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Over Expression, Knockdown

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: (A) Reverse transcription-quantitative PCR and (B) western blot analysis showing the downregulation of SOX11 and FAK at the mRNA and protein level in AT2 epithelial cells following CS. Data are presented as the mean ± standard error of the mean. * P<0.05. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: (A and B) Reverse transcription-quantitative PCR and (C-E) western blot analysis showing the effects of SOX11 overexpression and knockdown on the expressions of SOX11 and FAK at the mRNA and protein level in AT2 cells. Expression of SOX11 and FAK was significantly upregulated at both the mRNA and protein level following SOX11 overexpression. Knockdown of SOX11 decreased the levels of SOX11 and FAK. Data are presented as the means ± standard error of the mean. * P<0.05. AT2, alveolar type II; CS, cell stretch; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Knockdown, Expressing

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: (A-E) Western blot analysis showing Akt, MMP7 and p65 protein expression in AT2 epithelial cells. Akt expression increased significantly following SOX11 overexpression, and significantly decreased following knockdown of SOX11. No changes in MMP7 and P65 expression were observed. Data are presented as the means ± standard error of the mean. * P<0.05. AT2, alveolar type II; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase; MMP7, matrix metalloproteinase 7.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Western Blot, Expressing, Over Expression, Knockdown

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: (A-C) Western blot analysis showing caspase-3/8 expression in AT2 cells. The overexpression of SOX11 inhibited, whilst the knockdown of SOX11 increased caspase-3/8 expression. Furthermore, FAK antagonism blocked the effect of SOX11 overexpression on caspase-3/8 expression. Data are presented as the means ± standard error of the mean. * P<0.05, ** P<0.01. AT2, alveolar type II; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Western Blot, Expressing, Over Expression, Knockdown

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: Co-IP showing the interaction between SOX11 and FAK. CoIP results revealed that when (A) a SOX11 antibody and agarose beads were incubated with the AT2 cells lysates, SOX11 was detected in the bound protein, and its expression was markedly upregulated when SOX11 was overexpressed compared with the control group. When a FAK antibody was incubated with the bound protein, FAK bands were also detected in the bound protein, and the expression of FAK was markedly increased in the SOX11 overexpression group compared with the control group, similar to the expression of SOX11. (B) When AT2 cell lysates were incubated with FAK antibody and agarose beads, similar results were observed as that with the SOX11. The results suggested that SOX11 was bound with FAK and regulated the expression of FAK in AT2 cells. Input, band from the lysate of AT2 cells; IgG, negative control for the SOX11 or FAK antibody. AT2, alveolar type II; IP, immunoprecipitation; SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Co-Immunoprecipitation Assay, Incubation, Expressing, Control, Over Expression, Negative Control, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: SOX11 and FAK participate in the stretch-induced mechanical injury to alveolar type 2 epithelial cells

doi: 10.3892/ijmm.2020.4795

Figure Lengend Snippet: Luciferase assay showing the effects of SOX11 overexpression on luciferase activity and the expression of FAK mRNA in 293T cells in each group. It was shown that transfection with (C and E) pGL3-FAK promoter increased, while transfection with (B and E) pcDNA SOX11 had no effect on the luciferase activity compared with the vector group (A and E). (D and E) Following co-transfection with pcDNA SOX11 and pGL3 FAK promoter, the luciferase activity was further significantly increased compared with the vector group and pcDNA + pGL3-FAKP group. (F) RT-qPCR analysis revealed that the mRNA expression of FAK significantly increased in the pcDNA SOX11 + pGL3 and pcDNA-SOX11 + pGL3-FAKP groups. Data are presented as the means ± standard error. * P<0.05. SOX, Sex-determining gene on the Y chromosome related high mobility group box; FAK, focal adhesion kinase.

Article Snippet: AT2 cells (details provided below) were randomly divided into 5 groups as follows: i) The sham group, cells were cultured in normal culture medium for 72 h, corresponding to the culture time following transfection in the other groups, and then subjected to the sham treatments for CS; ii) CS group, AT2 cells were cultured for 72 h first and then underwent CS; iii) SOX11 overexpression group, AT2 cells were transfected with SOX11 plasmid and underwent CS 72 h following transfection with the SOX11 plasmid; iv) SOX11 plasmid + FAK antagonist group, AT2 cells were transfected with SOX11 plasmid and treated with 1.5 mM FAK antagonist (PF562271; cat. no. GC12174, Glpbio Technology, Inc., dissolved in DMSO) for 72 h and then subjected to CS; v) SOX11 siRNA group, AT2 cells were subjected to CS at 72 h following transfection with SOX11 siRNA.

Techniques: Luciferase, Over Expression, Activity Assay, Expressing, Transfection, Plasmid Preparation, Cotransfection, Quantitative RT-PCR