Journal: Cell death discovery
Article Title: Exploring the potential of selective FKBP51 inhibitors on melanoma: an investigation of their in vitro and in vivo effects.
doi: 10.1038/s41420-025-02430-y
Figure Lengend Snippet: Fig. 3 SAFits impair TGF-β expression and signaling. Western blot assay showing both the monomeric and dimeric form of TGF-β (a) and p- Smad2/3 (b) in A375 melanoma incubated in the absence or presence of 20 nM SAFit 1 or 30 nM SAFit 2. γ-Tubulin and total Smad were used as loading control. Full length western blots are shown as Supplemental Material. A densitometric analysis of bands was performed using ImageJ 1.42q for Macintosh. c Relative normalized expression values of TβRI mRNA levels in A375 melanoma cells incubated for 5 h in the presence or absence of 20 nM SAFit 1 or 30 nM SAFit2. Relative quantitation of the transcript was performed using co-amplified β-Actin as an internal control for normalization. Lower, Western blot of protein extracted from the same cells for TβRI assay. Full gels are shown as Supplemental Material. d Representative images (10x magnification fields containing ≥100 cells) of transwell migration and invasion assay of A375 melanoma cells cultured 48 h in the presence of 20 nM SAFit 1 or 30 nM SAFit2. Means of cell count values and crystal violet O.D. quantization obtained from different experiments (N = 3) are indicated below.
Article Snippet: Primary antibodies against the following proteins were diluted as follows: Flag (M2, mouse monoclonal, Merck) 1:5000; β-Actin, (15G5A11/E2, mouse monoclonal, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 1:5000; TGF-β (V; rabbit polyclonal, Santa Cruz Biotechnology) 1:500; γ-Tubulin (GTU-88, mouse monoclonal, Merck) 1:5000; phospho-Smad2 (Ser465/467, Cell Signaling Technology, Danvers, MA, USA) 1:500; Smad 2/3 (H465, rabbit polyclonal, Santa Cruz Fig. 5 SAFit-induced changes of TME composition in TAMs. a Representative flow cytometry gating of CD45+ cells infiltrating the tumors. b, c Graphic representation of cell count values from TME immunophenotyping (Tumor Infiltrating Leukocytes TILs: macrophages, B and T cells, NK) of SAFit untreated (white histograms) or treated (grey histograms) tumors. d Characterization of F4/80 macrophage component of the TME.
Techniques: Expressing, Western Blot, Incubation, Control, Quantitation Assay, Migration, Invasion Assay, Cell Culture, Cell Counting
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