Journal: Molecular and cellular endocrinology

Article Title: GDF15 inhibits early-stage adipocyte differentiation by enhancing HOP2 expression and suppressing C/EBPα expression.

doi: 10.1016/j.mce.2025.112461

Figure Lengend Snippet: Fig. 2. GDF15 expression is reduced by lysosomal degradation via the autophagic pathway. (A) Changes in GDF15 protein levels upon autophagic inhibition. ADSCs were treated with autophagy inhibitors, including chloroquine (CQ, 50 μM), ammonium chloride (NH4Cl, 25 mM), leupeptin (200 μM), pepstatin A (100 μM) + E−64 (35 μM), for 24 h prior to performing western blotting. (B, D) ADSCs were pre-treated with CQ (50 μM) for 1 h, and after changing the medium to a differentiation medium (DM), were cultured for 16 h and then fixed. Subsequently, the cells were treated with (B) antibodies against GDF15 (green) or SQSTM1 (red) and (D) antibodies against GDF15 (green) or LAMP1 (red) prior to capturing images using a confocal microscope. Yellow = merge/co-localization; scale bars: 10 μm. (C, E) Quantification of the co-localization between GDF15 and MAP1LC3B (C) and GDF15 and LAMP1 (E). Merged images in (B and D) were assessed for GDF15: MAP1LC3B (B) and GDF15:LAMP1 (D) co-localization using NIS Elements software and Pearson correlation analysis. Data are presented as the means ± standard deviation of 10 randomly selected cells for each treatment; ***P < 0.001. (F–H) Changes in GDF15 expression induced by knockdown of autophagy-related genes: ATG5 (F), ATG16L (G), and SQSTM1 (H). After transfection with scrambled siRNA or ATG5-, ATG16L-, and SQSTM1-specific siRNA in ADSCs, the medium was changed to DM and cultured 24 h later. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Images of the stained cells were acquired using a confocal laser microscope (A1 R HD25 NSIMS; Nikon, Tokyo, Japan) at the three-dimensional immune system imaging core facility of Ajou University.

Techniques: Expressing, Inhibition, Western Blot, Cell Culture, Microscopy, Software, Standard Deviation, Knockdown, Transfection