Fig 2: IKKβ participated in the translocation of SNAP23 to STX17‐contained autophagosomes mediated by TIP60. (A, B) RT‐qPCR and Western blot measured IKKβ expression. (C) RT‐qPCR assessed the knockdown efficiency of IKKβ. (D) Western blot evaluated the protein level of SNAP23. (E) Co‐IP examined the interaction between SNAP23 and STX17. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: MiR-34a confers chemosensitivity in BC cells. (A-D) The miR-34a stable cells were transfected with or without STX17 plasmid (1 µg/µl) for 24 h followed by exposing to various concentrations of chemotherapeutic drugs, including cisplatin (0-50 µM), doxorubicin (0-200 µg/ml), epirubicin (0-200 µM) and mitomycin C (0-200 nM). After 24 h of treatment, resazurin-based cell viability was measured. (E-H) The miR-34a stable cells were treated with chemotherapeutic drugs, including cisplatin (1.5 µM), doxorubicin (0.1 µg/ml), epirubicin (0.1 µM) and mitomycin C (12.5 nM) for 7 days. Cell survival was detected by colony formation assay. All data are expressed as the mean ± SDs in triplicate samples. *P<0.05, **P<0.01, and ****P<0.0001 relative to the control group. miR, microRNA; BC, bladder cancer; STX17, syntaxin 17.

Fig 4: STX17 is a downstream target of Xist-miR-23b-3p/miR-29a-3p axis and regulates PF formation.a The Venn diagram shows overlapping of the co-target genes of miR-23b-3p/miR-29a-3p from five representative databases. b The predicted binding sites of miR-23b-3p and miR-29a-3p binding sites in the 3′-UTR of Stx17. The red nucleotides represent mutant sequences of target sites. c Luciferase activities in HEK293 cells transfected with WT or mutant Stx17 plasmid together with miR-23b-3p or miR-29a-3p, or miR-NC. d Enrichment of Stx17 pulled down by biotin-miR-23b-3p, biotin-miR-29a-3, or biotin-miR-NC control in 3T3 cells. e–h Relative Stx17 mRNA (left), and STX17 protein (right) expression in cultured newborn ovaries transfected with mimics for miR-23b-3p, miR-29a-3p, or miR-NC (e), anti-miR-23b-3p, anti-miR-29a-3p, or anti-miR-NC (f), Xist or empty control (g), and siXist or scrambled control (h). i Relative Stx17 mRNA (left), and STX17 protein (right) expression at the indicated time points. j Relative Stx17 mRNA (left), and STX17 protein (right) expression in cultured newborn ovaries transfected with siStx17, or siNC as a control. k Representative images (left) and quantification of follicles (right) in newborn ovaries transfected with siStx17, or siNC. Scale bars: 50 μm. l qRT-PCR analysis of genes involved in PF survival and development in newborn ovaries transfected with siStx17, and siNC as a control. m WB analysis of LC3B expression in newborn ovaries treated under indicated condition. n Representative images of DDX4 in newborn ovaries co-transfected with pcDNA-Xist or empty vector together with siStx17 or scrambled control. Scale bars: 50 μm. o Quantification of follicles in n. p WB analysis of STX17 expression in newborn ovaries treated under indicated condition. GAPDH was used as a loading control. Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: YY1 is essential for Xist expression and functions as an upstream regulator for Xist-miR-23b-3p/miR-29a-3p-Stx17 axis during PF formation.a Schematic representation for YY1 binding motif within the proximal promoter region of Xist, with respective primer sets to amplify YY1 binding region. The upstream control region is also represented (top). Bottom: ChIP analysis of YY1 binding in newborn mouse ovaries at 0.5 dpp, and 4.5 dpp. The dashed line represents the basal IgG binding. b, c Relative Yy1 mRNA (b), and YY1 protein (c) expression in perinatal ovaries at indicted time. d, e Relative Yy1 mRNA (top), and YY1 protein (bottom) expression in cultured newborn ovaries transfected with siYy1 or control (d), pcDNA3.1-Yy1, and NC control (e). f Relative Xist mRNA expression in cultured newborn ovaries transfected with siYy1 or siRNA control (top), pcDNA3.1-Yy1 or NC control (bottom). g, h Representative DDX4 immunofluorescence images (g) and quantification of follicles (h) in newborn ovaries transfected under indicated condition. Scale bars: 50 μm. i WB analysis of STX17 expression in newborn ovaries treated under indicated condition. j Proposed model for Xist-miR-23b-3p/miR-29a-3p-STX17 axis in regulating oocyte loss in the perinatal mouse ovaries. Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001.