Fig 2: Intraurothelial macrophages phagocytose neutrophils and UPEC.a Confocal microscopy demonstrating phagocytosis of GFP+ UPEC (green) by F4/80+ macrophages (red) in the EpCAM-1+ urothelium (white) 1-day post-infection. b GFP+ UPEC in the urinary bladder determined by fluorescent image analysis (SCHNELL) after macrophage depletion by intraperitoneal application of aCSF1R antibodies. c The colony forming units of UPEC were determined in bladder homogenates three days post-infection. d Flow cytometry-based quantification of phagocytosis of neutrophils by macrophages (F4/80+Ly6G+) in bladder digests 1-day post-infection. e Confocal microscopy demonstrating phagocytosis of Ly6G+ neutrophils (green) by F4/80+ macrophages (red) in the EpCAM-1+ urothelium (white) 1-day post-infection. f Density of neutrophils in the urinary bladder after macrophage depletion by intraperitoneal application of aCSF1R antibodies 1-day post-infection. *p < 0.05, ***p < 0.001. Error bars show the mean ± SEM. lu=lumen. The scale bars in a, d indicate 5 µm and the white dashed lines distinguish the EpCAM-1+ urothelium from the lumen.

Fig 3: Macrophage relocation and the defense against UPEC depends on IL-6.Mice were infected with GFP-expressing UPEC and analyzed 1-day post-infection. IL-6 expression was topically inhibited by inoculating IL-6 antibodies (aIL-6) into the urinary bladder. Inoculating IgG antibodies served as the control. a Bladder tissue sections were stained with DAPI (blue), F4/80 (red) and EpCAM-1 (white) and the images were acquired by a fluorescence microscope. Dashed lines separate the urothelium from the connective tissue. The scale bars in the top row indicate 200 µm, bottom row 30 µm. b The density of F4/80+ macrophages in the bladder compartments was calculated by SCHNELL on immunofluorescent images. c The colony forming units of UPEC were determined in bladder homogenates in the presence and absence of IL-6. d Spider plots of the expression intensities [au] of the proteins of the GO terms “macrophage migration” (GO: 1905517) and “macrophage activation” (GO: 0042116) in the urothelium determined by MALDI-MSI. Asterisks inside the circle refer to the statistical analysis between healthy and UPEC infected (* in black), whereas the asterisks outside the spider plot (* in blue) compares UPEC versus aIL-6. *p < 0.05, **p < 0.01. Error bars show the mean ± SEM; lu, lumen.

Fig 4: Analysis of mouse pancreatic CTCs identifies Wnt2 as a candidate CTC genea, Schematic representation of strategy for DGE of RNA isolated from primary mouse pancreatic tumour, metastatic ascites, and CTC-enriched blood. b, Immunofluorescence staining of mouse pancreatic CTC, leukocyte (WBC), and CTC cluster captured on HbCTC-Chip (DAPI, blue; Cytokeratin (CK), red; CD45, green). c, Quantitation of CK+ cells captured from control mice and pancreatic cancer bearing mice (dashed line - threshold of = 6 CK+ cells/100 µL). d, DEGseq M-A plot of RNA sequence reads from mouse (MPANC-9) CTCs, comparing anti-EpCAM-captured versus IgG-coated HbCTC-Chips. (M - log2 fold change, A - averaged log2 reads, red dots – differentially expressed genes) e, RNA-ISH of CTCs and metastatic ascites cells co-expressing CK8+18 (green) and Wnt2 (red) transcripts. White arrowheads highlight Wnt2 signals. f, RNA-ISH of primary pancreatic tumours showing a small cluster of Wnt2 mRNA expressing cells near a pancreatic duct (D) (Wnt2, red dots; hematoxylin, light blue). Hematoxylin and eosin (H&E) stained serial section is shown (right). A second primary tumour imaged under fluorescence demonstrating Wnt2 expression in a subpopulation of tumour cells; high magnification shown (right, Wnt2, red; CK, green; DAPI, blue). (Scale bars=10 µm).

Fig 5: Overexpression of miR-449a induced TEPC differentiation into mature mTEC in vitro. (A,B) Immunofluoresence staining of TSC miR-449a cells and control cells with antibodies: EpCAM (G8.8), Aire (M-300) (A), K5 and K8 (B). (upper panel: TSC Control, lower panel: TSC miR-449a, white arrow in (A): Aire+, blue arrow in (A): Aire-,white arrow in (B): K5+K8-). (C) Flow cytometry analysis of TSC miR-449a cells and TSC Control cells with anti-Aire-Alexa 647 (5H12) and isotype control antibody. (D) Percent of Aire+ TSCs in cultures of TSC miR-449a cells and TSC Control cells from 5 randomly selected view fields of immunofluoresence images in (A). (E) Percent of K5+K8-TSCs in cultures of TSC miR-449a cells and TSC Control cells from 5 randomly selected view fields of immunofluoresence images in (B). (F) Immunoblot analysis of Aire, RelB, p52, DNMT3a and SATB2 in protein extracts of TSC miR-449a cells and TSC Control cells. GAPDH, Tubulin act as loading control. (G) RT-PCR analysis of indicated genes in TSC miR-449a cells and TSC Control cells, Tubulin acts as a loading control. (n ? 3).