trophoblasts in PE
To elucidate the molecular mechanisms underlying the autophagy of placental trophoblasts in early-onset PE, we employed RNA-seq to examine autophagy-related differentially expressed genes (DEGs) in placentas derived from early-onset PE pregnancies compared with those from non-PE pregnancies. The functionally related genes were enriched predominantly in autophagy-related signaling pathways, such as the PI3K-Akt signaling, AMPK signaling, and mTOR signaling pathways (Figure 1A ). The cluster heatmaps show the top 20 DEGs involved in autophagy-related signaling pathways in placentas from PE pregnancies (Figure 1B ). Among them, TP53BP2 was the most robust inducer (Figure 1C ). Moreover, TP53BP2 expression was markedly greater in placentas from PE pregnancies than in those from non-PE pregnancies(Figure 1D ). Immunohistochemical and immunofluorescence staining further revealed the upregulation of TP53BP2 in trophoblasts from PE pregnancies (Figure 1E, F) . To investigate the functional role of TP53BP2, we conducted TP53BP2 overexpression and knockdown in HTR8/Svneo and JEG-3 cells by transfecting Ad-TP53BP2 or three independent sh-TP53BP2 vectors, respectively (Figure S1 ). TEM revealed increased autophagosome and autolysosome formation in trophoblasts transfected with Ad-TP53BP2, whereas the opposite effect was observed in those transfected with sh-TP53BP2 (Figure 1G ). Additionally, an autophagic flux assay using tandem fluorescent GFP-RFP-LC3 revealed a decrease in total autophagosome and autolysosome formation in trophoblasts transfected with sh-TP53BP2 (Figure 1H ). LC3B-II and p62 serve as complementary markers of autophagy, with LC3B-II reflecting autophagosome formation and p62 indicating the process of substrate recognition and degradation. Therefore, the protein expression of LC3B-II and p62 was assayed to determine the extent of autophagy. The results of the western blot analysis confirmed the effect of TP53BP2 on the LC3B-II and p62 levels in HTR8/SVneo and JEG-3 cells under hypoxia (Figure 1I ). These results demonstrated that the upregulation of TP53BP2 could increase trophoblast autophagy in PE placentas.