Fig 2: AF9 Interacts with Snail and Hampers Snail Transcriptional Activity(A) Transcription factor (TF) enrichment of AF9 target genes demonstrated that most genes were potentially regulated by SNAI1 transcription factor, with q value less than 0.0001 considered statistically significant. Description of this method is provided in the Materials and Methods section. (B) Mass spectrometry analyses of AF9-associated proteins in MDA-MB-231 cells. The data are presented by a volcano plot. Proteins with a p value <0.0001 and Log2 (FLAG-AF9 versus FLAG) >2.0 were regarded as candidate proteins that significantly interact with AF9. The enlarged red point indicates Snail; other significant proteins are marked as green points. All significant proteins are listed in the larger circle. (C) In MDA-MB-231-FLAG-AF9 OE cells, M2 FLAG beads was used to immunoprecipitate the AF9 associated complex. Western blot analysis was performed using the indicated antibodies. (D) HA-Snail and FLAG-AF9 (WT or Y78A) were cotransfected into MDA-MB-231 cells. Antibody against FLAG was used to immune-precipitate the FLAG-AF9 associated complex. Western blot analysis was performed to test the interaction between Snail and AF9 using the indicated antibodies. (E) Separate deletion of three Snail regions (N-terminal, middle domain, and C-terminal), as shown in the upper panel. FLAG-AF9 and HA-Snail (including WT, dN ter, dM, and dC ter) were cotransfected into MDA-MB-231 cells. Antibody against HA was used to immunoprecipitate HA-Snail full length and truncated Snail associated complex. Ter, terminal; M, middle. (F) Snail mRNA expression and protein levels were tested in MDA-MB-231-Vehicle and MDA-MB-231-AF9 OE (WT or Y78A) cells. (G) Snail transcriptional activity was determined in MDA-MB-231-Vehicle and MDA-MB-231-AF9 OE (WT or Y78A) cells by a dual luciferase assay. These cells were cotransfected with the pGL3-CDH1 promoter (−57 to +149 relative to TSS) and Renilla control plasmid pRL-TK for 48 h. The relative luciferase activities were normalized to those of the cells expressing vehicle and then those of the Renilla control. The data represent the mean ± SD of three independent experiments. (H) ChIP assay was performed in MDA-MB-231-AF9 OE (WT or Y78A) cells with indicated the antibodies. Primers against the promoter regions of CDH1, CLDN3, VDR, and CXADR were used to perform quantitative real-time PCR to measure the binding affinity of Snail. The calculation of the relative binding affinities of Snail to the target regions is described in the methods section. (F–H) Two-tailed Student’s t test, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. Error bars represent the standard deviation (SD).

Fig 3: Analysis of human iPSC-BMELC barrier disruption based on RNA-seq. A Comprehensive RNA-seq was performed using total RNA with or without SARS-CoV-2 infection. Heatmap shows screened up- and downregulated genes by using cut-off values of fold change of ≥ 1.5 followed by second selection based on KEGG pathway analysis and references. B After SARS-CoV-2 infection for 24 h, tight junction marker genes (CLDN3, CLDN5, CLDN11, and CLDN12) were analyzed by RT-qPCR. C After SARS-CoV-2 infection for 24 h, expression of CLDN3 and CLDN11 was analyzed by western blot. Cropped blots were shown and the full-length blots were indicated in Fig. S5. The relative band densities were analyzed using ImageJ software and normalized to β-actin. Data are represented as mean ± SD (n = 3). *P < 0.05

Fig 4: Validation of HGSOC-specific EV markers.(A) Immunoblot analysis of three candidate proteins. FRα, Claudin-3, and TACSTD2 were validated in sEVs from four cell lines (cancer: CAOV3 and OVCAR3, and noncancer: HOSE1 and MTK), and TCLs were also tested. (B) Single-particle imaging measurement was performed using the ExoView platform. Representative images are shown at the top, and the number of detected sEVs is displayed as bar charts. CD9/CD63/CD81 were used as capturing antibodies on the chip and the fluorophore-conjugated antibodies against FRα (red), Claudin-3 (green), or TACSTD2 (blue) to label the targets. Scale bars, 1 μm. (C) Gene expressions [Log2(FC)] for FOLR1, CLDN3, TACSTD2, the genes of FRα, Claudin-3, or TACSTD2, obtained in pan-cancer analysis (n = 31 malignancy category).

Fig 5: TROP2− foetal HHyPs are restricted to the ductal plate of foetal liver. a RNA-ISH for CDH6 and STAT1 on human second trimester (15–21 pcw) foetal liver ductal plate (DP) and bile duct (BD) regions. Scale bars represent 50 μm. b Immunofluorescence (IF) staining of CDH6 (yellow), CLDN3 (magenta) and STAT1 (grey) co-expression in human foetal liver slides. Slides counterstained in DAPI (cyan). DP and BD structures outlined in white. Scale bars represent 25 μm. c Immunohistochemistry (IHC) of EpCAM, CK19, CDH6 and STAT1 in BD and DP regions of human foetal liver. Scale bars represent 50 μm. d Duplex RNA-ISH for CDH6(red)/STAT1(blue) and TROP-2(red)/STAT1(blue) in foetal liver BD and DP structures. Scale bars represent 50 μm. Scale bars of zoomed in region represent 25 μm. e Phase contrast imaging and IF of foetal intra-hepatic organoids (f-IHOs) derived from EpCAM enriched foetal liver cells in liver expansion (LE) media. All structures are counterstained with DAPI (blue). All staining performed between passages 3 and 5. f Schematic and IF staining of f-IHOs cultured for 7 days in hepatic differentiation (HD media) and biliary differentiation (BD media). All staining performed between passages 3 and 5. Images representative of n = 3 foetal liver differentiation experiments