Fig. 4

Hsa_circ_0002348 inhibited trophoblast proliferation and induced apoptosis through the repression of mTOR/ERK1/2 signaling pathway. A Representative images of TUNEL staining in the pregnant mice’s placentas of each group (n = 3/per group for each experiment); TUNEL signals in red, and DAPI-stained nuclei in blue; Scar bars, 50 μm; B qRT-PCR analysis of the expression levels of hsa_circ_0002348 in HTR8 cells following its knockdown by two distinct siRNAs; C qRT-PCR analysis of the expression levels of hsa_circ_0002348 in HTR8 cells with its overexpression vector; CCK-8 assay showing the proliferation ability of HTR8 cells after transfected with two siRNAs D and its overexpression vector E; colony-formation assays showing the proliferation capacity of HTR8 cells with two siRNAs F and hsa_circ_0002348 overexpression vector G; flow cytometry of fluorescence analysis stained with Annexin V-FITC and PI in HTR8 cells after hsa_circ_0002348 knockdown by two distinct siRNAs H and its overexpression vector I; the percentage of cells in the four different quadrants calculated, the results presented in different histograms indicating the fraction of the apoptotic cells as Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺; the quantitative results of F–I displayed in the right; J western blotting analysis detecting the expression level of p-mTOR, mTOR, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK and JNK when compared with Tubulin as control in HTR8 cells with NC or with two hsa_circ_0002348 siRNAs as treatment; the quantitative analysis of band intensity performed with Image J (right); circ_0002348 representing hsa_circ_0002348; NC negative control, Ctrl control; the data presented as mean ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001,****p ≤ 0.0001