zm306416hcl  (Tocris)

Journal: iScience

Article Title: Positive and negative feedback regulation of the TGF-β1 explains two equilibrium states in skin aging

doi: 10.1016/j.isci.2024.109708

Figure Lengend Snippet: Regulatory network of THBS1 and FMOD in skin aging (A) WB of THBS1 and FMOD in TGF-β1-stimulated HFF-1 cells. Cell lysates were collected 48 h after control (blue) or TGF-β1 (red) treatment. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively. (Left panel) Representative image. (Middle panel) Quantification of THBS1; N = 3, ∗ p < 0.05 (vs. control, Dunnett’s test). (Right panel) Quantification of FMOD; N = 3, ∗ p < 0.05, ∗∗ p < 0.01 (Dunnett’s test). (B) WB analysis of THBS1 in TGF-β1- and FMOD-stimulated HFF-1 cells. Cell lysates were collected 48 h after treatment with control (blue), 4 ng/mL TGF-β1 (red), or a combination of 4 ng/mL TGF-β1 and 8 ng/mL FMOD (purple). The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively.(Left panel) Representative image. (Right panel) Quantification of THBS1; N = 3, ∗ p < 0.05, ∗∗ p < 0.01 (Tukey’s multiple comparisons). (C) FMOD ELISA of THBS1-stimulated HFF-1 cells. Cells were treated with THBS1 (purple) for 48 h and their supernatants were analyzed. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively; N = 3, ∗ p < 0.01 (Dunnett’s test). (D) WB analysis of THBS1 in THBS1-stimulated BJ cells. Cell lysates were collected 48 h after control (blue) or THBS1 (purple) treatment. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively. (Left panel) Representative image. (Right panel) Quantification of THBS1; N = 3, ∗∗ p < 0.01 (Dunnett’s test). (E) WB analysis of THBS1 with c-Fos/c-Jun knockdown (KD) using HFF-1 cells. Cells were pretreated with each siRNA (50 nM) and collected 48 h after control (blue) or 4 ng/mL TGF-β1 (red) treatment. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively. (Left panel) Representative image. (Right panel) Quantification of THBS1; N = 3, ∗∗ p < 0.01 (Tukey’s multiple comparisons). (F) FMOD ELISA of kinase inhibitor-treated HFF-1 cells. Cells were treated with ZM306416HCl, Ki8751, GW5074, or U0126 for 48 h and their supernatants were analyzed. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively; N = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (Dunnett’s test). (G) FMOD ELISA of VEGF165 treated HFF-1 cells. Cells were treated with VEGF165 for 48 h and the supernatants were analyzed. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively; N = 3, ∗ p < 0.05 (Dunnett’s test). (H) FMOD ELISA in kinase inhibitor- and TGF-β1-treated HFF-1 cells. Cells were treated with Akt inhibitor VIII or LY294002 combined with 4 ng/mL TGF-β1 for 48 h and their supernatants were analyzed. The horizontal line in the center of the box plot is the median, the lower and upper borders indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values, respectively; N = 3, ∗∗ p < 0.01 (Student’s t test), † p < 0.05, †† p < 0.01 (Dunnett’s test). (I) Regulatory network of THBS1 and FMOD. In human dermal fibroblasts, TGF-β1 induced THBS1 production occurs via TGF-βR–SMAD activation (red shade). FMOD was regulated via activation of the VEGFR-cRaf-MEK pathway (blue shade). Crosstalk between these pathways occurred with the TGF-β pathway inhibiting the VEGF pathway via the PI3K-Akt pathway (green shade).

Article Snippet: ZM306416HCl , Tocris Bioscience , Cat# 3514-2499.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Knockdown, Activation Assay

Journal: iScience

Article Title: Positive and negative feedback regulation of the TGF-β1 explains two equilibrium states in skin aging

doi: 10.1016/j.isci.2024.109708

Figure Lengend Snippet:

Article Snippet: ZM306416HCl , Tocris Bioscience , Cat# 3514-2499.

Techniques: Recombinant, Modification, Western Blot, Blocking Assay, Electron Microscopy, Bicinchoninic Acid Protein Assay, Chromatin Immunoprecipitation, Purification, Isolation, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Control, Software