3D culture manuscripts supplemental data

<p dir="ltr"><a href="" target="_blank">Obesity poses a significant global health threat, particularly due to the </a>vital role of excessive visceral adipose tissue in metabolic dysfunction.<a href="" target="_blank"><i> In vitro</i>, the adipogenic potential of visceral adipose preadipocytes is markedly diminished in two-dimensional (2D) culture systems compared to their counterparts</a><i> </i><i>in vivo</i>. <a href="" target="_blank">This limitation drive</a>s the development of advanced culture systems that better replicate the adipogenesis <i>in vivo</i> environment. In this study, we established a novel three-dimensional (3D) spheroid culture model for visceral preadipocytes. Our findings indicate that this 3D model enhances the adipogenesis of epididymal white adipose tissue (eWAT) preadipocytes by inhibiting the Hippo signaling pathway and reducing the expression of downstream YAP protein. Notably, N⁶-methyladenosine (m⁶A), the predominant internal modification on mRNAs in eukaryotes, was significantly decreased in 3D cultured eWAT preadipocytes at day 1 post-treatment with the MDI cocktail. <a href="" target="_blank">Concurrently</a>, the expression of METTL3, the m⁶A methyltransferase, was markedly elevated in 3D cultured preadipocytes. In contrast, mRNAs of insulin-like growth factor 2 mRNA-binding proteins (<i>Igf2bps</i>), which encode RNA-binding proteins, showed a significant reduction in 2D monolayer cultured eWAT precursor cells, a pattern distinct from that of 3T3-L1 preadipocytes. The decreased levels of IGF2BPs may influence the m⁶A modification of critical genes within the Hippo signaling pathway, thereby facilitating eWAT preadipocytes adipogenesis. Collectively, these results demonstrate that 3D spheroid culture enhances visceral preadipocyte adipogenesis via modulation of the Hippo-YAP signaling pathway and reduces the m⁶A levels in 3D cultured visceral preadipocytes.</p>