Fig 1: FOXC2 is able to functionally substitute for FOXC1 during lymphatic valve development.(a) Schematic representation of the targeting vector and targeted allele. The entire protein coding region of Foxc1 is replaced with that of Foxc2. ACN, self-excision cassette including Cre driven by the testis-specific promoter. (b) Southern blot analysis to detect double-resistant ES cell colonies using 5’ and 3’ probes. (c) PCR genotyping of F1 heterozygotes to detect the Foxc1c2 allele. (d, e) Representative images of lymphatic valves in mesenteric collecting vessels immunostained with antibodies targeted to FOXC1 and VEGFR-3 from P12 P6 Foxc1+/+ (d) and Foxc1c2/c2 (e) mice. Scale bars are 25 µm. (f – h) Representative images of lymphatic valves in mesenteric collecting vessels immunostained with antibodies targeted to FOXC1, FOXC2, and VE-Cadherin from P6 Foxc1+/+ (f), Foxc1c2/+ (g), and Foxc1c2/c2 mice (h). Scale bars are 50 μm. (i – k) Representative images of the mesenteric vasculature immunostained with antibodies targeted to PROX1 and CD31 in P6 Foxc1+/+ (i), Foxc1c2/+ (j), and Foxc1c2/c2 mice (k). Scale bars are 200 μm. (l – n) Representative images of P6 mesenteric vasculature from P6 Foxc1+/+ (l), Foxc1c2/+ (m), and Foxc1c2/c2 mice (n) immunostained with antibodies targeted to FOXC1, FOXC2, and VE-Cadherin show gradual loss of FOXC1 expression in the blood and lymphatic vasculature and smooth muscle cells and conversely the increase of FOXC2 expression in blood vasculature and smooth muscle. Scale bars are 200 μm. (o) Quantification of total lymphatic valve number in lymphatic collecting vessels of P6 Foxc1+/+, Foxc1c2/+, and Foxc1c2/c2 individuals. N = 6 for Foxc1+/+, N = 8 for Foxc1c2/+, and N = 8 forFoxc1c2/c2 individuals. (p) Percentage of mature and immature lymphatic valves normalized to total valves counted in P6 Foxc1+/+, Foxc1c2/+, and Foxc1c2/c2 individuals. Data are presented as mean (± SD) and analyzed using Student’s t-test. NS denotes no significance.
Fig 2: Inducible deletion of aurora A in osteoblasts inhibits glucocorticoid-induced bone loss. (a) FACS analysis of demarrowed femurs of Col1a1cre/ERT2AuroraAflox/flox mice with tamoxifen and dexamethasone treatment using anti-VE-cadherin and anti-Osteocalcin antibodies; (b) Immunoblotting with densitometry analysis of aurora A, Foxc2, CD31, Osterix (OSX), and Osteopontin in Osteocalcin-positive cells isolated from demarrowed femurs of Col1a1cre/ERT2AuroraAflox/flox mice with tamoxifen and dexamethasone treatment. β-actin was used as loading control; (c,d) ChIP assay at glucocorticoid receptor DNA-binding site of the Foxc2 gene (c) and the Osterix gene (d) using anti-aurora A antibodies in Osteocalcin-positive cells isolated from demarrowed femurs of Col1a1cre/ERT2AuroraAflox/flox mice with tamoxifen and dexamethasone treatment (n = 8). (c,d) were analyzed for statistical significance using ANOVA with post hoc Tukey’s analysis. Error bars are maximal and minimal values. ***, p < 0.0001.
Fig 3: FENDRR increases FOXC2 expression by competitively binding miR-4700-3p (A) Prediction of the potential miRNAs targeting both FENDRR and FOXC2 with two independent miRNA target databases. (B) qRT-PCR analysis of miR-4700-3p expression in the indicated cells. (C) The expression of miR-4700-3p following the downregulation or upregulation of FENDRR expression in the indicated cells. (D) Western blot analysis of FOXC2 expression in SGC7901/ADR and SGC7901/VCR cells transfected with miR-4700-3p mimics or inhibitors and the corresponding negative controls. (E, F) Predicted potential binding sites of miR-4700-3p to FENDRR and FOXC2 are shown (upper panel). The wild-type (wt) and mutant (mut) miR-4700-3p target sequences of FENDRR (E) and FOXC2 (F) were fused to a luciferase reporter and cotransfected into HEK-293 cells with miR-4700-3p mimics or a negative control. Luciferase activity values were measured and normalized to empty vector control values. (G, H) Western blot analysis of FOXC2 expression in the indicated cells. **P < 0.01, *P < 0.05, N.S., not significant (P > 0.05), error bars, s.d.
Fig 4: miR-4700-3p suppresses drug resistance in GC (A, B) Survival of SGC7901/ADR and SGC7901/VCR cells transfected with miR-4700-3p mimics (A) and SGC7901 cells transfected with miR-4700-3p inhibitors or the corresponding negative controls was evaluated using the CCK-8 assay after step-up concentration of ADR and 5-FU treatment for 72 h. (C, D) The apoptotic rate of the indicated cells treated with 5-FU was shown. (E, F) SGC7901/ADR and SGC7901/VCR cells were cotransfected with miR-4700-3p, FOXC2 vector or their negative controls. Cell survival (E) and apoptosis rate (F) of the indicated cells treated with ADR or 5-FU, respectively, was evaluated. (G) Representative data extracted from TCGA datasets showing correlation between FENDRR (left) and FOXC2 (right) expression with miR-4700-3p expression in GC tissues (n=100). (H) Kaplan-Meier analysis of the correlation between miR-4700-3p expression and overall survival in GC patients included in the TCGA datasets (n=100). **P < 0.01, *P < 0.05, error bars, s.d.
Fig 5: Schematic model showing the role of the FOXC2/STC1/ITGB6 signaling axis in the regulation of metastasis, lipid metabolism and DDP chemoresistance in OC cells
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