Journal: Cell Communication and Signaling : CCS

Article Title: PSPC1-SMAD3 axis regulates iron-induced beiging of adipocytes in white adipose tissue

doi: 10.1186/s12964-025-02570-9

Figure Lengend Snippet: Iron inhibits the activation of SMAD3 in beige adipocytes. A Schematic diagram illustrating the protocol for ATAC-sequencing in in vitro differentiated beige adipocytes treated with FAC. B ATAC-seq signal distribution near the transcription start sites (TSS). C Pie chart showing the genomic distribution of all identified ATAC-seq peaks. D Motif enrichment analysis of differential ATAC-seq peaks (comparing FAC vs. CON). E Transcription factor footprint (TF) analysis for SMAD2::SMAD3 within differential ATAC-seq peaks. F Relative mRNA levels of Smad2 and Smad3 in CON and FAC-treated beige adipocytes. G , H Protein levels of phosphorylated and total SMAD2 and SMAD3 levels in CON and FAC-treated beige adipocytes. I , J Immunoblots phosphorylated SMAD3 (p-SMAD3), total SMAD3, UCP1, FABP4, and FTH1 in beige adipocytes treated with vehicle (CON), iron chelator deferoxamine (DFO), or DFO + FAC. Data in are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s., not significant

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h, followed by incubation overnight at 4 °C with the following primary antibody: anti-UCP1 (Abcam, ab234430), anti FTH1 (Abclone, A19544), anti phospho-SMAD3 (Ser423/425) (CST, #9520), anti SMAD3(CST, #9523), anti phospho-SMAD2 (Ser465/467), anti SMAD2 (ZENBIO, 382472), anti FABP4 (Proteintech, 12802-1-AP), anti PSPC1 (Proteintech, 16714-1-AP), anti HSP90(Proteintech, 60318-1-Ig), antiβ-Actin (Abclone, AC026), anti PGC1α (Proteintech, 66369-1-Ig).

Techniques: Activation Assay, Sequencing, In Vitro, Western Blot