Fig 1: a-b) Enrichment analysis of averaged 10% of most highly expressed genes (3437 genes) returns predominantly vascular-related terms. Over-representation analysis (ORA) using the enrichR package in R and the “Descartes Cell Types and Tissue 2021” database was used to identify gene sets that are statistically over-represented. The threshold value of enrichment was selected by a p-value <0.05, indicating that over-represented genes were significantly enriched for vascular-related terms. a) Count for genes in our dataset that are present in returned gene sets. b) Ratio for genes in our dataset that are present in returned gene sets, determined by the total number of genes in each set. c-i) Isolated microvessels have increased expression of canonical neurovascular-related genes. c) Bar plots showing TPMs for endothelial-defining genes, displayed according to general expression range. Highest expression found in endothelial genes B2M, BSG, FLT1, IFITM3, MT2A, SLC2A1, VIM, and VWF. d) Bar plot showing TPMs for pericyte-defining genes. Highest expression was detected in pericyte genes CALD1, FN1, IGFBP7, RGS5, and SPARCL1. e) Bar plot showing TPMs for smooth muscle cell-defining genes. Highest expression was detected in smooth muscle genes ACTG1, ACTN4, MYL6, PTMA, and TAGLN. f) Bar plot showing TPMs for tight junction-defining genes and g) Bar plot showing TPMs for adherens junction-defining genes. Several genes encoding for junctional proteins are found in the top 10% of most highly expressed genes, including CLDN5, CTNNB1, CTNND1, OCLN, JAM1, TJP1, and TJP2. h) Bar plot showing TPMs for astrocyte-defining genes. Astrocytic gene expression was predominantly limited to markers of astrocytic processes or endfeet, namely CLU, GFAP, and GLUL. i) TPMs from neurovascular-related genes were summarized according to cell type expression, demonstrating an overrepresentation of endothelial, smooth muscle cell, and pericyte genes. j) Overlap between top 10% of most highly expressed genes from our RNA sequencing data and immune genes found within the Immunome Database. Bar plots were generated using the ggplot package and Venn diagram was generated using the ggVennDiagram package in R.
Fig 2: Detection of viral load in serum and brain and description of brain histopathological damage after infection of SV129 and SV129 (Vim-KO) mice with dengue virus (DENV)-2. (A, B) Changes in the viral load in the brain and serum; *P < 0.05; **P < 0.01; ***P < 0.001. (C, D) Brain histopathological section on the 5th day after infection in SV129 and SV129 (Vim-KO) mice. (C) Left: the cortical stratifications disappeared, with a large number of apoptotic pyknotic cells (black arrow). Right: the hippocampus displayed apoptotic pyknosis of glial cells (black arrow) and local cerebral liquefactive necrosis (black circled). (D) Left: the cortical stratification was normal, and apoptotic pyknosis of glial cells (black arrow) was found in all six cortical layers. Right: the hippocampus (black square) showed no obvious abnormalities.
Fig 3: Impact of RAD21 deficiency on NCC activity(A) Ki67 exhibited 0.4-fold lower in the siRAD21 group compared to the siCon group. n = 3.(B) During the NCC differentiation process, SA-β-Gal staining revealed enhanced senescence features in NCCs with RAD21 deficiency. Scale bar = 1370 μm. n = 3.(C) During NCC differentiation into keratocytes, RAD21 knockdown cells exhibited clustering. Scale bar = 200 μm. n = 3.(D) Immunofluorescence staining at the end of differentiation revealed lower expression of KERA in the siRAD21 group compared to the siCon group, while VIM was more enriched in the siRAD21 group than in the siCon group. The siRAD21 group displayed reduced cell numbers and clustered growth. Scale bar = 200 μm.(E) qPCR displayed consistent expression trends for KERA and VIM as observed in (D). The scleral marker MGP exhibited 38.9-fold higher in the siRAD21 group compared to the siCon group according to RNA sequencing data. Data are presented as mean ± standard deviation. n = 3. ∗p < 0.05, ∗∗∗p < 0.001.
Fig 4: The CD44s/ZEB1 feedback loop enables the miR-200/ZEB1 circuit to function as a three-way switch. (a) Top panel: The SNAIL-driven miR-200/ZEB1 circuit including the CD44s/ZEB1 feedback loop. Bottom panel: Bifurcation diagram of ZEB1 mRNA levels for the miR-200/ZEB circuit in response to SNAIL. (b) Top panel: The S 1-S 2-driven miR-200/ZEB1 circuit including the CD44s/ZEB1 feedback loop. S 1 represents a transcriptional inhibition signal on miR-200 and S 2 represents a transcriptional activation signal on ZEB1. Bottom panel: The phase diagram (a two-parameter bifurcation) of the miR-200/ZEB1 circuit driven by signals S 1 and S 2. In (a) and (b), the dotted bar-headed arrows represent the alternative splicing of CD44 mRNAs by ESRP1. Different colors in the bifurcation diagrams represent different co-existences of stable states. For example, the blue colored region marks the tristable phase – {E, E/M, M}, where all three phenotypes - E, E/M and M can be the stable states. In (b), the region marked by black dots in phase diagram represents the parameter region of S 1and S 2 for the existence of the hybrid E/M phenotype – existing either alone - {E/M} or in combination with other stable states – {E, E/M}, {M, E/M} and {E, E/M, M}. (c) Top panel: Relative gene expression levels of ZEB1 and ESRP1 in epithelial (n = 11), hybrid E/M (n = 11) and mesenchymal (n = 37) cell lines from NCI-60. Bottom panel: Pearson’s correlations between gene expression of ESRP1 and ZEB1, VIM, CDH1 and OVOL2. ‘*’ represents P value ≤0.05. ‘**’ represents P value <0.005. ‘***’ represents P value <0.0001. (d) Immunofluorescence images showing different expression patterns of EMT markers in NSCLC cell lines. In the first column, blue is for DAPI, red is for ZEB1 and green is for CDH1. In the second column, blue is for DAPI, red is for CDH1 and green is for VIM. (e) mRNA levels of CDH1, VIM, SNAIL and ZEB1 in NSCLC cell lines. (f) Protein levels of CDH1, VIM, SNAIL, ZEB1 and FOXC2 in NSCLC cell lines. In (e) and (f), H820 and H1437 are epithelial cell lines, H1299 and H2030 are mesenchymal cell lines, H1975 is hybrid E/M cell line and H1944 is a mixture of E and M cells
Fig 5: ADAM9 can activate the expression of CDH2 in aggressive lung adenocarcinoma cell lines.(A) Quantitative RT-PCR of CDH2 in the aggressive cell line BM7 and its control line, CL1-0; 18S rRNA was used as a loading control. **, P<0.005. (B) Western blot analysis of ADAM9 and CDH2 in BM7 and CL1-0 cells. L: long form of ADAM9; S: short form of ADAM9. EF1A was used as a loading control. EF1A: elongation factor 1 alpha. (C) Relative expression levels of CDH2 in BM7 cells transfected with two siRNAs against ADAM9. Two primer sets (I and II) targeting different CDH2 regions were used to amplify the CDH2 products. Two short hairpin RNAs targeted against ADAM9 (shADAM9-C & shADAM9-E) were examined. HPRT was used as a loading control. *, P<0.05. (D) Western blot analysis of CDH2 in the ADAM9-depleted BM7 cells. EF1A was used as a loading control. CDH1: E-cadherin; VIM: vimentin. (E) Immunohistochemistry analysis of ADAM9 and CDH2 in the ADAM9-depleted cells. Scale bar: 20 μm. (F) Western blot analysis of CDH2 in parental cells over-expressing ADAM9. ACTB was used as a loading control.
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