Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: Effect of BTXA on the viability of mouse L929 fibroblasts. (A) The morphological changes of fibroblasts observed under a microscope. (B) The vimentin identification of fibroblasts with Hoechst 33258 was determined by immunofluorescence assay. (C) BTXA inhibited fibroblast viability, which was detected by cell counting kit-8 assay in a dose- (0, 0.125, 0.25, 0.5, 1 and 2 IU/ml) and time- (12, 24 and 48 h) dependent manner. ** P<0.01 vs. control. BTXA, botulinum toxin type A.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Microscopy, Immunofluorescence, Cell Counting, Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: BTXA suppresses fibroblast viability and promotes apoptosis. (A) BTXA suppressed the high fibroblast viability induced by TGF-β1. (B) BTXA increased the apoptosis of fibroblasts induced by 10 ng/ml of TGF-β1. Dotted line separation represents whether or not fibroblasts were treated with TGF-β1. Data are shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^ P<0.05 and ^^ P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: BTXA prevents extracellular matrix (ECM) over-deposition in fibroblasts. BTXA decreased the high expression levels of collagen I, collagen III and α-SMA induced by 10 ng/ml of TGF-β1 in fibroblasts. The mRNA and protein levels of collagen I, collagen III and α-SMA were detected by (A) RT-qPCR and (B) western blot analysis, respectively. (C) α-SMA expression was determined by immunofluorescence. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblast were treated with TGF-β1. Data were shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^ P<0.05 and ^^ P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: BTXA suppresses the expression levels of MMP-2 and MMP-9. BTXA decreased the high expression levels of MMP-2 and MMP-9 induced by 10 ng/ml of TGF-β1 in fibroblasts. The mRNA and protein levels of MMP-2 and MMP-9 were respectively detected by (A) RT-qPCR and (B) western blot analysis, respectively. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblast were treated with TGF-β1. Data are shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^ P<0.05 and ^^ P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1; MMP, matrix metalloproteinase.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: Effect of BTXA on PTEN and caspase-3 expression, as well as PTEN methylation. (A) Effect of treatment of fibroblasts, which were cultured with or without 10 ng/ml of TGF-β1, with various concentrations (0.25, 0.5 and 1 UI/ml) of BTXA on the mRNA expression of PTEN, as determined by RT-qPCR. (B) Effects of BTXA on the protein levels of PTEN, pro-caspase-3 and cleaved-caspase-3, as detected by western blot analysis. (C) PTEN methylation was measured by methylation-specific PCR (MSP). M, methylated; U, unmethylated; MC, methylated control; UC, unmethylated control. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblast were treated with TGF-β1. Data are shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^ P<0.05 and ^^ P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1; PTEN, phosphatase and tensin homolog deleted on chromosome ten.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Expressing, Methylation, Cell Culture, Quantitative RT-PCR, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: BTXA inhibits the activities of DNMTs. (A) The high DNMT activity induced by 10 ng/ml of TGF-β1 was decreased by BTXA. (B and C) The mRNA and protein levels of DNMT1, DNMT3a and DNMT3b were determined by RT-qPCR and western blot analysis, respectively. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblasts were treated with TGF-β1. Data are shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^ P<0.05 and ^^ P<0.01 vs. control with TGF-β1. BTXA, botulinum toxin type A; TGF-β1, transforming growth factor-β1; DNMT, DNA methyltransferase.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Botulinum toxin type A prevents the phenotypic transformation of fibroblasts induced by TGF-β1 via the PTEN/PI3K/Akt signaling pathway

doi: 10.3892/ijmm.2019.4226

Figure Lengend Snippet: BTXA inactivates the phosphorylation of PI3K and Akt. (A) The protein expression of p-PI3K, PI3K, p-Akt and Akt was examined by western blot analysis. (B) BTXA notably suppressed the ratio of p-PI3K/PI3K. (C) BTXA notably suppressed the ratio of p-Akt/Akt. β-actin was used as an internal control. Dotted line separation represents whether or not fibroblasts were treated with TGF-β1. Data are shown as the means ± SD, n=3. * P<0.05 and ** P<0.01 vs. control without TGF-β1; ^^ P<0.01 vs. control with TGF-β1.

Article Snippet: After being blocked in bovine serum albumin (BSA), the fibroblasts were incubated first with anti-α-SMA antibody (A03744, 1:200; BosterBio) overnight at 4°C and then with Cy3-conjugated goat anti-rabbit secondary antibodies (BA1032, 1:500; Beyotime) for 0.5 h. Subsequently, the fibroblasts were stained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime) and images were captured under a fluorescence microscopy (Nikon).

Techniques: Phospho-proteomics, Expressing, Western Blot, Control

Journal: BMC Neurology

Article Title: Nuclear entrapment and extracellular depletion of PCOLCE is associated with muscle degeneration in oculopharyngeal muscular dystrophy

doi: 10.1186/1471-2377-13-70

Figure Lengend Snippet: PCOLCE expression levels are down-regulated in OPMD patients and in a muscle cell model system. A . Plot shows expression levels of PCOLCE in biopsies from pre-symptomatic muscle that carry expPABPN1 but show no disease symptoms (N = 4) and OPMD symptomatic samples (N = 5). Fold change was calculated by reference to age-matched controls (N = 19). Only in the symptomatic group PCOLCE is significantly deregulated (*p < 0.05). B . RT-qPCR analysis of Pcolce mRNA levels in TA muscles of wild-type (FVB) and A17.1 OPMD model mice at 26-weeks of age. Fold change was calculated after normalisation to the Hprt housekeeping gene. Average scores are from 6 mice. Asterisk indicates significant decline (*p < 0.05). C . Pcolce levels in myotube cultures expressing expPABPN1-FLAG (Ala17) at a level similar to endogenous Pabpn1. (i) RT-qPCR analysis of Pcolce mRNA expression in myotube cultures of IM2 parental and Ala17 transgene containing cells. Fold change was calculated after normalisation to the Hprt housekeeping gene mRNA. Variations between fusion efficiencies were eliminated after normalisation to expression levels from the muscle specific actin ( Acta1 ) gene. Averages are from 3 biological replicates. Asterisk indicates significant decline (*p < 0.05). (ii) Western blot analysis of soluble protein extracts from unfused (-) and 4-day fused (+) IM2 and Ala17 cultures. The blot was incubated with anti-Pcolce antibody. Human PABPN1 is detected with an anti-FLAG antibody, the ACTA1 (MSA) protein is a marker for myotube differentiation and tubulin is used as a loading control.

Article Snippet: Primary antibodies used were: mouse monoclonal anti-FLAG M2 (Sigma-Aldrich); mouse monoclonal anti-muscle actin (MSA) (Novocastra, Newcastle upon Tyne, UK); mouse monoclonal anti-α-Tubulin (Sigma-Aldrich); mouse anti-ubiquitin, FK2, (AbCam, Cambridge, UK); rabbit polyclonal anti-Pcolce (AbCam and Sigma-Aldrich); mouse anti-Dysferlin (Novocastra); anti-MBNL1 (donated by Dr. T.A.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Incubation, Marker