Journal: bioRxiv

Article Title: Testosterone exposure during fetal masculinization programming window determines the kidney size in adult mice

doi: 10.1101/2025.02.11.637631

Figure Lengend Snippet: (A) Anogenital distance of 3-month-old KO and WT males without and with testosterone supplementation (res) at E14.5-17.5. The lines indicate means. * = P ≤ 0.05 ** = P ≤ 0.01. (B) kidney weights of 3-month-old KO and WT males without and with testosterone supplementation (res) at E14.5-17.5. The lines indicate means. * = P ≤ 0.05 ** = P ≤ 0.01. (C) Igfbp5 expression (RNA-seq) in E16.5 kidneys of WT, KO and treated KO (res) groups (RPKM = Reads Per Kilobase Million). The lines indicate means. * = P ≤ 0.05. (D) Igfbp5 mRNA expression (qPCR) at different age points in WT kidneys. Points indicate means and whiskers SD. d = day, wk = week. (E) Immunofluorescent staining of IGFBP5 (red) and LRP2 (green) in 2-week-old WT kidneys. Examples of IGFBP5 staining are marked with red arrows. Scale bar: 100 μm. (F) Visualization of AR and HNF4A binding peaks near Igfbp5 in ChIP-seq data. T = testosterone treatment. (G) Proliferation analysis of HEK 239 cells cultured with 0 (Ctrl), 25, 50 or 100 nM recombinant human IGFBP5. The lines indicate means. * = P ≤ 0.05. (H) Top 10 most significant canonical pathways obtained from Ingenuity Pathway Analysis (IPA) of DE genes that were normalized in Hsd17b3 -/- kidney upon fetal androgen supplementation. The top x-axis represents the -log (P-value). (I) Representative western blot images in E15.5 and quantification of phosphorylated AKT (pAKT), AKT and loading control β-actin in E15.5 and E18.5 WT and KO kidney homogenates. The lines indicate means. (J) Representative western blot images in E15.5 and quantification of phosphorylated S6 ribosomal protein (pS6) and loading control β-actin in E15.5 and E18.5 WT and KO kidney homogenates. The lines indicate means. ** = P ≤ 0.01.

Article Snippet: For proliferation analysis, HEK 293 (ATCC) cells were cultured on poly-L-lysine –coated plates (#P4832, Sigma-Aldrich) and switched to serum-free medium (DMEM/F12, #D2906, Sigma; 1% Penicillin/streptomycin, #A5256701, Gibco; 1% L-Glutamine, #25030-024, Gibco; 20 nM DHT) for 3 hours before changing to fresh serum-free medium with 25, 50 or 100 nM recombinant human IGFBP5 (#875-B5, R&D Systems).

Techniques: Expressing, RNA Sequencing Assay, Staining, Binding Assay, ChIP-sequencing, Cell Culture, Recombinant, Western Blot, Control