Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT

doi: 10.1186/s13046-018-0995-9

Figure Lengend Snippet: MiR-214 inhibited the tumor-promoting ability of CAFs by directly targeting FGF9. a Ten predicted genes as potential targets of miR-214 were analyzed by qRT-PCR. Five of them were overexpressed in CAFs compared to NFs, in which FGF9 represented the most prominent one. b The protein expression levels of FGF9 in NFs and CAFs. c and d The migration and invasion abilities of cultured GC cells were suppressed after adding FGF9 neutralizing antibody into CAF-CM. e Among five upregulated genes as mentioned above, only FGF9 mRNA expression was reduced in CAF miR-214 compared to CAF NC . f FGF9 protein expression in CAFs was also suppressed in CAF miR-214 compared to CAF NC . g Two predicted binding sites between miR-214 and FGF9 3’UTR. h The relative luciferase activity was significantly suppressed in cells co-transfected with wild-type binding site vectors of FGF9 3’UTR in the presence of pre-miR-214 (* P < 0.05, ** P < 0.01)

Article Snippet: After incubation with bovine serum albumin for 20 min at room temperature, the slides were incubated with primary antibody FGF9 (1:150, R&D systems) overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining.

Techniques: Quantitative RT-PCR, Expressing, Migration, Cell Culture, Binding Assay, Luciferase, Activity Assay, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT

doi: 10.1186/s13046-018-0995-9

Figure Lengend Snippet: Correlation between FGF9 expression and clinicopathological parameters of gastric adenocarcinomas

Article Snippet: After incubation with bovine serum albumin for 20 min at room temperature, the slides were incubated with primary antibody FGF9 (1:150, R&D systems) overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT

doi: 10.1186/s13046-018-0995-9

Figure Lengend Snippet: FGF9 expression in primary tumor ( a – d ) and lymph node metastatic sites ( e – h ) of GC. a The high expression of FGF9 in CAFs, but low expression in tumor cells. b CAFs and tumor cells were both positive for FGF9. c The high expression of FGF9 in tumor cells, but low staining in CAFs. d CAFs and tumor cells were both negative for FGF9. e and f FGF9 staining was high in CAFs but low in tumor cells of both intestinal type ( e ) and diffuse type ( f ). g and h CAFs and tumor cells were both positive for FGF9 ( g , intestinal type and h , diffuse type). i and j The proportion of high expressed FGF9 in CAFs and tumor cells of primary tumor ( i ) and lymph node metastatic sites ( j ). The percentage of high expressed FGF9 in LNCAFs ( k ) and LNT ( l ) of intestinal-type and diffused and mixed-type

Article Snippet: After incubation with bovine serum albumin for 20 min at room temperature, the slides were incubated with primary antibody FGF9 (1:150, R&D systems) overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining.

Techniques: Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT

doi: 10.1186/s13046-018-0995-9

Figure Lengend Snippet: High FGF9 expression in lymph node metastatic sites CAFs was associated with poor prognosis in patients with GC. a – c The Kaplan–Meier survival analysis revealed that the FGF9 level in primary tumor CAFs, primary tumor cells, and lymph node metastatic sites tumor cells was not associated with prognosis. d High FGF9 expression in lymph node metastatic site CAFs was significantly associated with poor prognosis in patients with GC

Article Snippet: After incubation with bovine serum albumin for 20 min at room temperature, the slides were incubated with primary antibody FGF9 (1:150, R&D systems) overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining.

Techniques: Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Downregulation of miRNA-214 in cancer-associated fibroblasts contributes to migration and invasion of gastric cancer cells through targeting FGF9 and inducing EMT

doi: 10.1186/s13046-018-0995-9

Figure Lengend Snippet: High FGF9 level in lymph node metastatic site CAFs and tumor cells were associated with poor prognosis in diffuse and mixed–type GC. a and b High FGF9 level in lymph node metastatic site CAFs or tumor cells were not associated with prognosis in intestinal-type GC. c and d High FGF9 level in lymph node metastatic CAFs or tumor cells was associated with poor prognosis in diffuse and mixed–type GC

Article Snippet: After incubation with bovine serum albumin for 20 min at room temperature, the slides were incubated with primary antibody FGF9 (1:150, R&D systems) overnight at 4 °C and then with HRP-conjugated secondary antibody at room temperature for 30 min. DAB was used for staining.

Techniques:

Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: ( A ) Quantification of scratch wound closure of E13.5 growth-arrested primary dermal fibroblasts treated with FGF9 (200 ng/ml) or FGF9 and SU5402 (20 µM). At 24 hr, FGF9 treatment resulted in greater wound-closure at 8 hr (36.17 ± 5.04%, p=0.0490) and at 24 hr (97.11 ± 3.09%, p=0.0133) compared to DMSO control (all treatments n = 5 experiments, each performed with freshly extracted primary dermal fibroblast cell population). Wound closure was not altered when cells were treated with both FGF9 and SU5402 inhibitor (p=0.2563). ( B ) Quantification of transwell migration assay of E13.5 primary dermal fibroblasts. Migration was significantly increased when FGF9 (200 ng/ml) was added to lower or both upper and lower chambers (p=0.0003 and p=0.0010, respectively; for both n = 6 experiments, each performed with freshly extracted primary dermal fibroblast cell population). No statistical difference was observed between FGF9 treatments (p=0.4033). ( C ) Quantification of nuclei density in E13.5 dermis explants treated for 3 hr with beads loaded with FGF9 (100 µg/ml) or 0.1% BSA vehicle control. Density measured from a single optical slice at mid-bead. FGF9 bead induces an increase in density within 15 µm radius from the bead relative to BSA control (p=0.033, n = 7 explants), but not between 15 and 30 µm radius (p=0.236, n = 7 explants). ( D ) Whole-mount RNA in situ hybridization of dermal samples treated with FGF9 beads for 3, 8, and 16 hr. Induction of Spry4 and Dusp6 , but not Sox2 expression (purple) was observed around the bead at all time points ( n indicates induction/total samples, induction was tested in two independent experiments with skin samples derived from at least two different litters.). ( E ) 2 hr EdU incorporation into dermis organ cultures after overnight incubation with BSA (left), FGF20 (center), FGF9 (right) loaded beads. Note the increased number of proliferating cells around the FGF9 bead ( n = 5 explants). Error bars represent SD. *, p≤0.05; **, p≤0.01; ***, p≤0.001. Scale bar = 30 µm. See also and . 10.7554/eLife.36468.027 Figure 6—figure supplement 1—source data 1. Values used to quantify FGF9-induced cellular changes. Values used to quantify fibroblast wound closure in the presence of DMSO, SU5402, FGF9 +DMSO, or FGF9 +SU5402 . Values used to quantify E13.5 primary fibroblast transwell migration in control, FGF9 in lower chamber, and FGF9 in seeding and lower chambers . Values used to quantify fibroblast density in response to BSA or FGF9-loaded beads at 0–15 µm and 15–30 µm distance from the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Control, Transwell Migration Assay, Migration, RNA In Situ Hybridization, Expressing, Derivative Assay, Incubation

Journal: eLife

Article Title: Hair follicle dermal condensation forms via Fgf20 primed cell cycle exit, cell motility, and aggregation

doi: 10.7554/eLife.36468

Figure Lengend Snippet: 3 hr incubation of BSA-, FGF20- or FGF9-loaded beads with E13.5 wildtype ( A, B, C ) or Fucci mKO ( D ) dermises. Cdkn1a expression was assayed in these samples using ( A ) whole-mount RNA in situ hybridization of dermal samples. Cdkn1a expression (purple) was not observed around the bead (0/20 each condition, in four independent experiments with skin samples derived from four different litters.) ( B ) section radioactive in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters), and ( C ) section in situ hybridization (0/5 each condition in two independent experiments with skin samples from two different litters). ( D ) Cell cycle exit was assessed using Fucci mKO reporter allele (representing G 0 /G 1 cell cycle phase) in the 30 µm surrounding the center of the bead (0/10 each condition in two independent experiments with skin samples from two different litters). ( E ) Quantification of percent total cells surrounding the bead positive for Fucci-mKO . No significant difference was observed between any of the groups. Error bars represent SD. Scale bar = 50 µm. See also and . 10.7554/eLife.36468.029 Figure 6—figure supplement 2—source data 1. Values used to quantify FGF20 or FGF9 induced Fucci-mKO expression. Values used to quantify the percent of total cells expressing Fucci-mKO 30 µm surrounding the center of the bead .

Article Snippet: Peptide, recombinant protein , FGF9 human recombinant protein , R and D Systems , 273-F9 , .

Techniques: Incubation, Expressing, RNA In Situ Hybridization, Derivative Assay, In Situ Hybridization

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Establishment of a transgenic mouse model with the conditional deletion of Fgf9 in osteoblasts. A) Schematic showing the transgenic mouse model used in this study. B). The Cre recombinase is demonstrated to be expressed in osteoblasts (OBs) and osteocytes (OCTs) in adult Coll (2.3)-Cre; td/Tomato Red mice. C) Reverse transcription end point PCR demonstrated a successful excision of Fgf9 fragment in the RNA extract from the long bones of the Coll (2.3); Fgf9 fl/fl (Fgf9 OB−/−) mice. D) Real time PCR assessment of the level of Fgf9 mRNA (relative to GAPDH) in the long bones of 3 months old Fgf9 OB−/− mice. *p < 0.05 vs. littermate controls (Fgf9 fl/fl).

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Transgenic Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of cancellous bone mass at distal femur in the 3 month old male and female Fgf9 OB−/− mice. A). Representative μCT 3D reconstruction images of the distal femurs. B and C). Histomorphometry of cancellous bone at the distal femurs. BV, bone volume; TV, tissue volume; Tb.Th, trabecular thickness, Tb.Sp, trabecular separation; MS, mineralizing surface; MAR, mineral apposition rate, BFR, bone formation rate. ** p < 0.01, *p < 0.05 vs. the sex-matched littermate controls. ### p < 0.001, ## p < 0.01 vs. age-match Fgf9 fl/fl mice with the same genotype.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: μCT assessment of distal femur and tibiofibular junction in 3 month of Fgf9 OB−/− and littermate Fgf9 fl/fl mice.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of cortical bone at tibio-fibular junction (TFJ) in the 3 month old male and female Fgf9 OB−/− mice. A). Representative 3D reconstruction images of μCT scan at the TFJ. B and C). Histomorphometry of cortical bone at the TFJ. Ps.Pm, periosteal perimeter; Ec.Pm, endosteal perimeter; Ct.Th, cortical thickness; Med.Ar, bone marrow area; Ps.BFR, periosteal bone formation rate, Ec.BFR, endosteal bone formation rate. ** p < 0.01, *p < 0.05 vs. the sex-matched littermate controls.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Determination of mRNA levels of osteogenic marker genes in the long bones of the 3 month old Fgf9 OB−/− mice. *p < 0.05 vs. the sex-matched littermate controls.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Marker

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Assessment of bone resorption in the 3 months old Fgf9 OB−/− mice. N. OC, number of osteoclasts; BS, the length of bone surface. PYD, pyridinoline.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques:

Journal: Bone

Article Title: Osteoblast-derived FGF9 regulates skeletal homeostasis

doi: 10.1016/j.bone.2016.12.005

Figure Lengend Snippet: Effects of exogenous FGF9 on proliferation of the cultured bone marrow stromal cells (BMSCs). The BMSCs from wild type mouse were treated with exogenous FGF (5 ng/ml) for 24 h. A and B) Assessment of BMSC proliferation in the absence or presence of Akt inhibitor, MK-2206 (1 μM). Cell proliferation was determined by BrdU incorporation assay. C) Determination of Akt1 activation in BMSCs by FGF9 (5 ng/ml) using Western blots.

Article Snippet: On day 3, 1–10 ng/ml recombinant mouse FGF9 (R&D Systems, INC, Minneapolis, MN) was added to the media for 24 h, and BrdU was added 4 h prior to the assay.

Techniques: Cell Culture, BrdU Incorporation Assay, Activation Assay, Western Blot

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 1 FGF9 increases total neuronal length in Q7 and Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 24 and 48 h and then fixed for immunofluorescent staining using a βIII tubulin antibody. Immunofluorescent images show neuronal morphology in Q7 (a) and Q111 (b) cells at 24 and 48 h after FGF9 treatment. βIIItubulin: green color. Hoechst 33342: blue color. Quantitation results in the immunofluorescent images provide a comparison of total outgrowth in the Q7 (c) and Q111 (d) cells. Double asterisks represent p < 0.01, triple asterisks represent p < 0.001, N = 45–103 cells from three different batches

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Cell Culture, Staining, Quantitation Assay, Comparison

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 2 FGF9 increases expression levels of neuronal morphology–related proteins in Q7 and Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 48 h, and then subjected to western blotting. Western blotting was performed in Q7 (a) and Q111 (c) cells using βIII tubulin,

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Molecular neurobiology

Article Title: Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

doi: 10.1007/s12035-020-02220-w

Figure Lengend Snippet: Fig. 3 FGF9 increases expression levels of neuronal synaptic markers in Q111 cells. Q7 and Q111 cells were cultured with or without FGF9 for 48 h and then subjected to western blotting. Western blotting was performed in the Q7 (a) and Q111 (c) cells using synaptophysin and PSD-95 antibodies. γ-tubulin was used as an internal control. Quantitation results after western blotting pro- vide a comparison of these markers in the Q7 (b) and Q111 (d) cells. Triple asterisks represent p < 0.001. N = 6

Article Snippet: For the FGF9 treatments, the cells were cultured in medium with 10% FBS for 24 h, and then the medium was replaced with a serum-free medium with or without FGF9 recombinant proteins (50 ng/ml; R&D Systems) for 24 or 48 h. To inhibit NF-kB signaling, the cells were pretreated with BAY11-7082 (1 μM; InvivoGen) 1 h before FGF9 treatment and then cultured for 48 h. The cells subjected to different treatments were collected for further examination.

Techniques: Expressing, Cell Culture, Western Blot, Control, Quantitation Assay, Comparison