Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: Expression of S100 family members is associated with EMT, and mesenchymal S100 proteins stimulate invasion of PDAC cells. a Immunoblot analysis of EMT-TFs, EMT markers and S100 proteins in a panel of PDAC cell lines. b Analysis of the transcription of ZEB1-regulated S100 genes in epithelial PDAC cells. BxPC-3 and SU.86.86 cell lines were transfected with the plasmid vectors expressing GFP-tagged ZEB1 or GFP control and cultured for 48 h. Bar charts show the expression of genes encoding S100 proteins and EMT markers analysed by qPCR. Data represent the mean of three replicate experiments ± StDev. c Invasion of mesenchymal and epithelial PDAC cell lines in zebrafish embryos. Cells were fluorescently DilC12-labelled (red), microinjected into the perivitelline cavities of zebrafish embryos, imaged and analysed 48 hpi as described in Methods. Merged phase contrast and fluorescence images are representative examples of zebrafish embryos with no invasion (upper image) or with PDAC cells that intravasate into the circulation (lower image). Minimum 10 fish per cell line were used in each experiment. Results are mean ± StDev of three independent experiments. d Mesenchymal S100 proteins, S100A4 and S100A6, contribute to the enhanced invasive potential of PDAC cells in vivo. siRNA-mediated knockdowns of mesenchymal S100 genes reduce the invasion of AsPC-1 and MIA PaCa-2 cells in zebrafish assay. Western blots show the extent of depletion of S100A4 and S100A6. Results are means ( n = 3 biological replicates; 10 fish in each experiment) ± StDev. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Control, Cell Culture, Fluorescence, In Vivo

Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: IL-11/STAT3 and ZEB1 regulate expression of S100 proteins in a mutually independent mode. a Treatment with IL-11 does not induce an EMT in epithelial PDAC cells. BxPC-3 and SU.86.86 cells were treated with IL-11 or mock-treated and the expression of EMT-TFs and EMT markers was analysed by immunoblotting as indicated. b Ectopic expression of ZEB proteins does not affect STAT3 phosphorylation. Effect of ZEB proteins on S100A4, S100A6 and pSTAT3 expression levels was analysed in indicated epithelial cell lines. Note that ZEB proteins regulate S100A4 and S100A6 independently of STAT3. ( c ) Combined effects of ZEB1 and IL-11 on the expression of S100 proteins. ZEB1-expressing or non-expressing cells were treated with IL-11 or left untreated, and level of the indicated S100 proteins was analysed by immunoblotting. d Schematic representation of EMT-ZEB1 and IL-11/6-STAT3 pathways regulating expression of mesenchymal (S100A4 and S100A6) and an epithelial (S100A14) S100 proteins in PDAC cells. ZEB1 cooperates with the inflammatory signalling to induce S100A4 and S100A6. On the other hand, it abolishes an effect of IL-11/IL-6 on the expression of S100A14. Reduced expression of S100A14 further stimulates production of S100A4 and S100A6. This regulatory network drives the invasion of PDAC cells

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: S100 proteins are expressed in pancreatic lesions. a Examples of IHC analyses of the expression of S100 proteins in a series of PDAC samples ( n = 31). b Images exemplify co-expression of mesenchymal S100 proteins and E-caherin in parallel sections of a PDAC sample. c Bar charts illustrate the frequency of the expression of S100A4 and S100A6 proteins in different types of pancreatic lesions presented on TMA

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: Association between EMT markers and expression of S100 proteins in PDAC samples

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: IL-6/11-STAT3 signalling regulates expression of S100 proteins in PDAC cells. a IL-6 and IL-11 enhance expression levels of S100A4 and S100A6 proteins in PDAC cell lines. PDAC cells were exposed to the specified cytokines (200 ng/ml) for 48 h, and changes in protein expression levels were assessed by immunoblotting with indicated antibodies. b IL-11 enhances expression of S100 proteins belonging to both mesenchymal and epithelial groups. BxPC-3 and SU.86.86 cells were cultured in the presence of IL-11 for 48 h, and protein expression was analysed by immunoblotting. c STAT3 mediates the effect of IL-11 on the expression of S100 proteins in epithelial PDAC cell lines. Expression of S100 proteins was stimulated by IL-11 treatment for 48 h. A selective STAT3 inhibitor stattic was added 24 h before cells were harvested, and expression of S100 proteins was analysed by immunoblotting. d STAT3 controls expression of S100 proteins in mesenchymal cells, where their expression is intrinsically high. MIA PaCa-2 cells were treated with stattic for 24 h prior the immunoblot analysis with indicated antibodies was carried out. e Parallel sections from the same block containing a PDAC tissue were analysed by IHC. The images exemplify samples, in which S100A4, S100A6 and pSTAT3 proteins are co-expressed

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Expressing, Western Blot, Cell Culture, Blocking Assay

Journal: British Journal of Cancer

Article Title: ZEB1 and IL-6/11-STAT3 signalling cooperate to define invasive potential of pancreatic cancer cells via differential regulation of the expression of S100 proteins

doi: 10.1038/s41416-019-0483-9

Figure Lengend Snippet: Mesenchymal S100 proteins mediate IL-11/STAT3-driven invasion in zebrafish embryos. a IL-11 stimulates invasion of PDAC cells in STAT3-dependent manner. Epithelial PDAC cells were treated with the control siRNA, or siRNA targeting S100A4, S100A6, or STAT3, cultured in the presence or absence of IL-11 for 48 h, and analysed in zebrafish invasion assay. Expression of STAT3 and S100 proteins was analysed by immunoblotting. b BxPC-3 and SU.86.86 cells were treated with IL-11 alone or in combination with stattic, and their invasiveness was assessed in zebrafish embryos. c Mesenchymal MIA PaCa-2 cells were treated with stattic, or mock-treated, and invasion analysed in zebrafish embryos. a – c Results are mean ± StDev of biological replicates ( n = 3). Cell invasion was analysed in 10 fish in each experiment. * p < 0.05; ** p < 0.01; *** p < 0.001 (Student’s t -Test)

Article Snippet: We used the following primary antibodies raised against ZEB1 and S100A8, (both from Santa Cruz Biotechnology, Dallas, USA); S100A4 and S100A6 (both from Proteintech, Manchester, UK); S100A2, S100A9, S100A14, S100P, Tubulin and β-Actin (all from Sigma Aldrich); SNAIL1, SNAIL2, STAT3, pSTAT3, p65 (Cell Signaling Technology, Danvers, MA, USA); E-cadherin and P-cadherin (BD Bioscience, San Jose, CA, USA); S100A11 (R&D Systems, Minneapolis, MN, USA); TWIST1 (Abcam Cambridge, MA, USA); ZEB2 (in-house made ).

Techniques: Control, Cell Culture, Invasion Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Amyloid-beta deposition and reduced drainage at the cribriform plate lymphatics in APP/PS1 mouse model of Alzheimer’s Disease

doi: 10.1101/2025.05.15.654007

Figure Lengend Snippet: Anatomical scheme of the olfactory bulb (OB) - cribriform plate (CP) region from the dorsal view. Note the outward projecting olfactory nerve bundles (ON) through the CP into the nasal epithelium (NE) (A). Position “B” (dotted line) in the coronal view displays clear skull foramen where circular cross sectioned ONs transverse CP (B). Whole-head decalcified image (C) of CP foramina region of 24-month APP/PS1 mouse stained with for LYVE-1 (ALY7), CD45 (30-F11), Amyloid-beta (D54D2), and DAPI with zoomed in image (D) which shows Aβ in CP foramina and perineural space of ON. Anatomical scheme (E) where position “ F ” (dotted line) in the coronal view displays the rostral edge of OB and ON which have transversed the underside CP (F). Whole-head decalcified image of rostral CP region of 24-month-old APP/PS1 mouse (G) stained with for LYVE- 1 (ALY7), CD45 (30-F11), Amyloid-beta (D54D2), and DAPI with zoomed in images (H- I) which show Aβ in perineural space of ON. OB = olfactory bulbs, ON = olfactory nerves, NE = Nasal Epithelium, CP = Cribriform Plate, and BM = Bone Marrow.

Article Snippet: Sections were stained with rabbit anti-amyloid-beta (1:100, D54D2, Cell Signaling Technology), rat anti-LYVE-1-eFluor-660 conjugated (1:200, ALY7, 50-044-82, Thermo Fisher Scientific), rat anti-Podoplanin-eFluor488 conjugated (1:500, 53-5381-80, eBioscience), rat anti-CD45-AlexaFluor488 (1:200, 30-F11, Invitrogen, 53-0451-82), and Methoxy X04 (1:1000).

Techniques: Staining

Journal: bioRxiv

Article Title: Amyloid-beta deposition and reduced drainage at the cribriform plate lymphatics in APP/PS1 mouse model of Alzheimer’s Disease

doi: 10.1101/2025.05.15.654007

Figure Lengend Snippet: Healthy 24-month-old control (A) and 24-month-old APP/PS1 mice (B) were perfused and tissues were collected for whole-head decalcification. Sections were stained for amyloid-β (D54D2) and LYVE-1 (ALY7). 60X image showing close association of amyloid-β within the lymphatic vessels (C). Decalcified whole-head section of 24-month APP/PS1 mice were stained for DAPI and amyloid-β (4G8) (D). Decalcified whole-head section of 7-month-old 5XFAD was stained with Congo Red and Hematoxylin and imaged under polarized light microscopy (E) with magnified images shown (F). Quantification of amyloid-β in lymphatic area (G). OB = olfactory bulbs. Representative image of n=4-5 mice. Results of a Student’s unpaired t-test, p = 0.002.

Article Snippet: Sections were stained with rabbit anti-amyloid-beta (1:100, D54D2, Cell Signaling Technology), rat anti-LYVE-1-eFluor-660 conjugated (1:200, ALY7, 50-044-82, Thermo Fisher Scientific), rat anti-Podoplanin-eFluor488 conjugated (1:500, 53-5381-80, eBioscience), rat anti-CD45-AlexaFluor488 (1:200, 30-F11, Invitrogen, 53-0451-82), and Methoxy X04 (1:1000).

Techniques: Control, Staining, Light Microscopy

Journal: bioRxiv

Article Title: Amyloid-beta deposition and reduced drainage at the cribriform plate lymphatics in APP/PS1 mouse model of Alzheimer’s Disease

doi: 10.1101/2025.05.15.654007

Figure Lengend Snippet: Healthy 24-month-old control (A) and 24-month-old APP/PS1 (B) were euthanized and perfused prior to tissue collection and whole-head decalcification. Sections were stained for CD45 (30-F11), LYVE-1 (ALY7), DAPI (A) and Amyloid-β (D54D2) (B). Several areas of CD45+ cells accumulating near LYVE-1+ vessels were shown (C). Close association of Amyloid-β and LYVE-1 was seen (D). Lymphatic vessel area and structure is altered in APP/PS1 mice (F) when compared to healthy (E) mice. LYVE-1+ vessel area and CD45+ cells of the shown images (A and B) were analyzed on ImageJ and results are shown (G and H). Three serial sections were taken of healthy and APP/PS1 mice and LYVE-1+ vessel area was measured. CD45+ cells at the LYVE-1+ vessel area (as shown in E and F) were counted in the three serial sections using Cell Counter on ImageJ. A Student’s unpaired t-test was performed on GraphPad Prism with p = 0.2684 (G) and p = 0.2139 (H). There is a trend of decreased LYVE-1 vessel area (G) and increased CD45+ immune cell recruitment (H).

Article Snippet: Sections were stained with rabbit anti-amyloid-beta (1:100, D54D2, Cell Signaling Technology), rat anti-LYVE-1-eFluor-660 conjugated (1:200, ALY7, 50-044-82, Thermo Fisher Scientific), rat anti-Podoplanin-eFluor488 conjugated (1:500, 53-5381-80, eBioscience), rat anti-CD45-AlexaFluor488 (1:200, 30-F11, Invitrogen, 53-0451-82), and Methoxy X04 (1:1000).

Techniques: Control, Staining