Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4 expression in SHF cells is required to maintain expression of key SHF-related proteins. (A-B‴) Transverse sections were evaluated for Mef2c and Fgf8 transcript expression at E9 in control (A) and Mef2c-AHF-Cre,Dll4F/F mutant (B) by RNAscope. Higher magnification of the boxed areas in A and B are shown as Mef2c expression (A′,B′), Fgf8 expression (A″,B″) and merged images (A‴,B‴) to demonstrate the reduced expression of Fgf8 transcripts in the mutants compared with the controls in the pharyngeal mesoderm (PM). (C-D‴) Similarly, transverse sections were evaluated for Mef2c and Fgf10 transcript expression at E9 in control (C) and Mef2c-AHF-Cre,Dll4F/F mutant (D). Higher magnification of the boxed areas in C and D are shown as Mef2c expression (C′,D′), Fgf10 expression (C″,D″) and merged images (C‴,D‴) to demonstrate that the PM in mutants has decreased expression of Fgf10 transcripts. (E-F‴) Transverse sections of control (E) and Mef2c-AHF-Cre,Dll4F/F mutant (F) E9.5 embryos were co-stained for Islet1 and Fgf8 protein expression. Higher magnification of the boxed areas in E and F are shown as Islet1 expression (E′,F′), Fgf8 expression (E″,F″) and merged images (E‴,F‴), showing reduced expression of Fgf8 in the SHF region. (G-H″) Transverse sections of control (G) and Mef2c-AHF-Cre,Dll4F/F mutant (H) E11.5 embryos were stained for Hand2 protein expression. Higher magnification of the boxed areas in G and H show the RV and LV in control (G′,G″) and mutant (H′,H″) embryos. Hand2 expression is lost in the mutant RV compared with controls. There is no change in the low basal level expression seen in LV. Scale bars: 50 µm (E′-F‴); 100 µm (A′-D‴,E,F,G′-H″); 250 µm (A-D,G-H).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Control, Mutagenesis, RNAscope, Staining

Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4-mediated notch signaling regulates Fgf8 expression in SHF. (A) Schematic of the mouse chromosome 10 around the region of the Fgf8 gene (E, exon). Putative RBPjk binding sites are indicated with asterisks. Constructs cloned for luciferase assay are shown as black boxes. Promoter 3 and Enhancer 2 were used as negative controls. (B) 293T cells were treated with DAPT to quench basal Notch activity. They were then transfected with various luciferase expression vectors with (empty bars) or without (solid bars) the NICD expression vector. Luciferase activity was measured in triplicate wells (mean±s.e.m.) 24 h later with eight experimental repeats. (C) The experiment was then repeated in triplicate after mutating the putative RBPjk binding site of Promoter 1. Mutation of putative binding sites led to loss of luciferase activity. (D-F″) Thoracic regions were dissected in control (D-D″) and Mef2c-AHF-Cre,Dll4F/F mutant (E-F″) embryos at E9.5 and cultured in vitro. Mutant organs were cultured with (F-F″) or without (E-E″) exogenous recombinant Fgf8. Sections were then co-stained for Islet1 and pHH3 expression to study SHF proliferation. Representative images are shown as Islet1 expression (D′,E′,F′), pHH3 expression (D″,E″,F″) and merged images (D,E,F). (G) Double-positive cells were counted in multiple fields (23 untreated control, 23 Fgf8 100 ng/µl control, 40 Fgf8 500 ng/µl control, 37 Fgf8-untreated mutant, 7 Fgf8 100 ng/µL mutant and 14 Fgf8 500 ng/µl mutant sections; mean±s.e.m.) showing a significant reduction in SHF proliferation in mutant organs compared with control (P<0.0001 between Fgf8-untreated control and mutant, P>0.05 between Fgf8-untreated controls and Fgf8-treated mutants by two-tailed t-tests). For quantification purposes, the boxed regions in D′, E′ and F′ were used as the area occupied by SHF progenitor cells. Exogenous Fgf8 supplementation had no significant impact on control embryos, but fully rescued proliferation defects seen in mutant embryos. (H-L′) Compound heterozygotes were analyzed by H&E staining of transverse sections of E14.5 embryos to demonstrate genetic synergy between Dll4-mediated Notch and Fgf8 signaling in SHF maturation. Cre-negative control embryos showed fully septated ventricles (H) and an aortic valve normally aligned over the left ventricle (H′). Heterozygous knockdown of Dll4 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt) also demonstrated a normal phenotype (I,I′). Heterozygous knockdown of Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Fgf8F/wt) showed a low incomplete penetrance of cardiac defects. The majority of the embryos showed a normal phenotype (J,J′). A shallow VSD (arrow in K) and a slightly mal-aligned aorta mildly over-riding the ventricular septum (arrowhead in K′) was seen in 14% of the embryos. Double heterozygous knockdown of Dll4 and Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt,Fgf8F/wt) showed high penetrance of DORV, with 83% of the embryos studied showing VSD (arrow in L) and a prominent over-riding of aorta with greater than 50% aorta arising from the RV (arrowhead in L′). (M) Table indicates number and phenotypes of embryos recovered amongst the different genotypes shown. The number of embryos recovered, the percentage recovery and the expected percentage recovery are based on Mendelian inheritance. Scale bars: 50 µm (D-F″); 300 µm (H-L′).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Binding Assay, Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Control, Cell Culture, In Vitro, Recombinant, Staining, Two Tailed Test, Negative Control, Knockdown

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: QRT-PCR (A) and imunoblot (B) analysis of FGF8 level in human CRC tissues (T) and adjacent normal mucosa tissues (N) from the same patient. All data were from at least three independent experiments. *, P<0.05; **, P<0.01.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Quantitative RT-PCR

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Immunohistochemical staining of FGF8 in tumor and corresponding colorectal mucosa. (B) Immunohistochemical scores for FGF8 in normal colorectal mucosa and CRC tissues. (C) Expression of FGF8 in the primary tumors without (N0) or with (N1/N2) lymph node metastasis was analyzed. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. (D) Kaplan–Meier survival curves of CRC patients with low (n = 34) and high (n = 38) FGF8 expression. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: RKO cells were treated with or without FGF8 and/or PD173074. (A) Representative photographs of colony formation 14 days after culture of cells. (B) Proliferation rate as measured by BrdU labeling for 12 hours. (C) Quantitative analysis of cell migration and matrigel invasion assays. Migration was analyzed at 24 h, and invasion at 48 h. (D) Representative phase-contrast images of RKO cell morphology. (E) Expression of Snail, E-cadherin and Vimentin was examined by immunoblot. All data were from at least three independent experiments. **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Labeling, Migration, Expressing, Western Blot

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Mean tumor volume and Ki67 expression in tumors after subcutaneous transplantation of RKO-FGF8 or RKO-mock cells. (B) Histopathology showing the lung metastases in mice and quantification of the lung weight and number of metastases following tail-vein injection with RKO-FGF8 or RKO-mock cells. **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Expressing, Transplantation Assay, Histopathology, Injection

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) We identified hub proteins implicated in core pathways according to the following four gold standards: degree, link, module and microarray analyses. (B) The global PPI network. (C) The core sub-network in cancer. (D) Identification of FGF8-YAP1 interaction in CRC cells.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Microarray

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Immunoblot analysis of cytoplasmic and nuclear YAP1 in RKO cells treated with or without FGF8 or/and PD173074. β-Actin was used as a cytoplasmic protein loading control, and histone-3 (H3) was used for nuclear protein loading control. (B) MRNA level CTGF and CYR61 was examined by qRT-PCR. (C) Transcription activity of TEAD4 was examined by luciferase assay. (D) Expression of YAP1 and FGF8 in serial human colorectal tumor sections was examined by immunohistochemical staining. (E) Correlation between the expression levels of YAP1and FGF8. (F) Expression of YAP1 in high-FGF8-expressing tumors and low-FGF8-expressing tumors was analyzed. All data were from at least three independent experiments.*, P<0.05; **, P<0.01.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Western Blot, Control, Quantitative RT-PCR, Activity Assay, Luciferase, Expressing, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: FGF8-treated RKO cells were transfected with siYAP1 or siNC. (A-B) Proliferative activity was measured by a colony formation assay (A) and BrdU labeling (B). (C) Cell migration and invasion was examined by transwell assay and matrigel invasion assays. (D) Representative phase-contrast images of RKO cell morphology. (E) Expression of Snail, E-cadherin and Vimentin was examined by immunoblot. All data were from at least three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Transfection, Activity Assay, Colony Assay, Labeling, Migration, Transwell Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: QRT-PCR analysis of YAP1 mRNA level in FGF8-treated RKO, SW480 and HCT116 cells in the present or absent of PD173074.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Quantitative RT-PCR