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: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 1. Inducible, cardiac-specific FGF9 trans- genic mice. A, Schematic representation of the binary transgene system. B, Time schedule of doxycycline (Dox) treatment and FGF9 transgene expression (blue, FGF9 not expressed; white, FGF9 expressed). C, Immunoblots showing FGF9 expression in the LV myocardium at 8 weeks of age (mice still on Dox) and at 10 and 16 weeks of age (mice off Dox for 2 or 8 weeks) in WT, tTA sin- gle transgenic, and tTA/FGF9 DTG mice from lines 1 and 3. D, Quantification of FGF9 protein expres- sion (8 to 10 animals per group).

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Expressing, Western Blot, Transgenic Assay

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 2. FGF9 promotes cardiac hyper- trophy. LV mass (A) and ratio of LV mass to body mass (B) in WT, tTA single transgenic, and tTA/FGF9 DTG mice 2 and 8 weeks after doxycycline (Dox) withdrawal. Hearts (C) and transverse heart sections (D) stained with Masson trichrome 8 weeks after Dox withdrawal. E, LV tissue sections stained with WGA and DAPI or Masson trichrome 8 weeks after Dox removal (scale bar, 100 m). F, Mean LV cardiomyocyte cross-sectional area (CSA) 2 and 8 weeks after Dox withdrawal; 8 animals per group in A, B, and F.

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Transgenic Assay, Staining

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 3. FGF9 enhances microvessel density in the myocardium. A, LV tissue sections from WT, tTA single transgenic, and tTA/FGF9 DTG mice stained with anti-Ki67 antibody and isolectin B4 to detect proliferating endothelial cells (top row), with isolectin B4 to detect capillar- ies (cardiomyocyte borders highlighted by WGA staining; middle row), and with anti-SMA antibody to detect conduc- tance vessels (cardiomyocyte borders highlighted by WGA staining; bottom row) 8 weeks after doxycycline (Dox) withdrawal (scale bars, 50 m). Quantifi- cation of Ki67 isolectin B4 cell density (B), capillary density (C), and conduc- tance vessel (D; 20 to 50 m) density in LV myocardium 2 and 8 weeks after Dox withdrawal (4 to 6 animals per group).

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Transgenic Assay, Staining

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 4. FGF9 stimulates endothelial cell proliferation, network formation, and release of prohypertrophic factor(s). A, Cell size of neonatal cardiomyocytes stimulated for 24 hours with 100 nmol/L endothelin 1 (ET1) or increasing concen- trations of FGF9 (3 experiments; *P0.05, ***P0.001 vs control). Prolifer- ation (B) and network formation (C) of HCAECs stimulated for 24 hours with 10 ng/mL vascular endothelial growth factor (VEGF) or increasing concentrations of FGF9 (3 to 5 experiments; *P0.05, **P0.01 vs control). D, Images of HCAECs under control conditions and after stimulation with 10 ng/mL FGF9. E, Images of neonatal cardiomyocytes kept for 24 hours under control conditions or stimulated with 100 nmol/L ET1, 10 ng/mL FGF9, supernatant (1:2 dilution) obtained from HCAECs kept for 24 hours in serum-free medium (control HCAEC-SN), or supernatant (1:2 dilution) obtained from HCAECs stimulated for 24 hours with 10 ng/mL FGF9 (FGF9 HCAEC-SN); cells were stained with an anti–troponin T antibody and DAPI. Cell size (F) and protein content (G) of neo- natal cardiomyocytes kept for 24 hours under the conditions described in E (4 to 6 experiments).

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Control, Staining

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 5. BMP6 is induced by FGF9 in vitro and in vivo and stimulates cardiomyocyte hypertrophy. A, Cell size of neonatal cardiomyo- cytes stimulated for 24 hours with supernatant (1:2 dilution) obtained from HCAECs kept for 24 hours in serum-free medium or stimu- lated for 24 hours with 10 ng/mL FGF9 (control and FGF9 HCAEC-SN). When indicated, a BMP6-neutralizing antibody or control anti- body (20 g/mL each) was added to the FGF9 HCAEC-SN. Cardiomyocytes were also stimulated with increasing concentrations of BMP6 in the presence or absence of the ALK2/3/6 inhibitor dorsomorphin (DM; 20 mol/L) or the ALK4/5/7 inhibitor SB-431542 (SB; 20 mol/L) (3 experiments). B, Immunoblot analysis of phospho-SMAD1/5 and phospho-SMAD3 expression in neonatal cardiomyo- cytes in response to stimulation with 10 ng/mL BMP6 for the indicated times (data are representative of 3 experiments). Cross- sectional area (CSA; C) and [14C] phenylalanine incorporation (E) of adult cardiomyocytes stimulated for 24 hours with FGF9, BMP6, or 10% FCS (75 to 111 cells per condition in C; 4 to 5 experiments in E; **P0.01, ***P0.001 vs control). D, Images of adult cardiomyo- cytes stimulated for 24 hours with 300 ng/mL FGF9, 10 ng/mL BMP6, or 10% FCS. F, Immunoblot analysis of BMP6, phospho- SMAD1/5, and phospho-SMAD3 expression in the LVs of WT, tTA single transgenic, and tTA/FGF9 DTG mice 8 weeks after doxycy- cline withdrawal (similar results were obtained in 2 additional mice per genotype).

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: In Vitro, In Vivo, Control, Western Blot, Expressing, Transgenic Assay

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 6. FGF9 reduces interstitial fibrosis and reduces heart failure mortality after MI. A, Immunoblot analysis of endogenous FGF9 expression in the noninfarcted LV myocardium of WT mice at various time points after LAD ligation (similar results were obtained in 2 additional mice per time point). B through E, Mice underwent LAD ligation 2 weeks after doxycycline withdrawal; end points were assessed 6 weeks later. B, Transverse sections of infarcted WT, tTA single transgenic, and tTA/FGF9 DTG hearts stained with Masson trichrome. C, Tissue sections from WT, tTA, and DTG hearts stained with Picrosirius Red and viewed under nonpolarized (top row) or polarized light (bottom row); images are from the non- infarcted part of the LV (scale bar, 200 m). D, Quantification of interstitial collagen vol- ume fraction in the noninfarcted LV myocar- dium (4 to 6 animals per group). E, Thirty- nine WT, 41 tTA, and 35 DTG mice were followed up for 6 weeks after LAD ligation.

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Western Blot, Expressing, Ligation, Transgenic Assay, Staining

Journal: Circulation

Article Title: Conditional Transgenic Expression of Fibroblast Growth Factor 9 in the Adult Mouse Heart Reduces Heart Failure Mortality After Myocardial Infarction

doi: 10.1161/circulationaha.110.989665

Figure Lengend Snippet: Figure 7. Adenoviral FGF9 gene transfer improves systolic function and reduces heart failure mortality after MI. WT mice underwent LAD ligation followed imme- diately by a single injection of Ad.lacZ or Ad.FGF9 into the LV cavity (1108 pfu each). A, Immunoblot analysis of FGF9 expression in the noninfarcted LV myo- cardium, liver, and lung 7 days after gene transfer. B, Time course of FGF9 protein expression in the noninfarcted LV after FGF9 gene transfer. C, LV tissue sections stained with WGA and an anti- FGF9 antibody 7 days after Ad.lacZ or Ad.FGF9 treatment (images are from the noninfarcted LV). D, LV mass and ratio of LV mass to body mass 6 weeks after LAD ligation in 12 Ad.lacZ- and 14 Ad.FGF9-treated mice. E, LV end-dia- stolic area (LVEDA), LV end-systolic area (LVESA), and LV fractional area change 6 weeks after LAD ligation in 10 Ad.lacZ- and 10 Ad.FGF9-treated mice (*P0.05, **P0.01 vs Ad.lacZ). F, Thirty Ad.lacZ- and 30 Ad.FGF9-treated mice were fol- lowed up for 6 weeks after LAD ligation.

Article Snippet: Antibodies against mouse FGF9 (Genway Biotech, San Diego, CA), BMP6 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-SMAD1/5, phospho-SMAD3, and β-actin (Cell Signaling Technology, Danvers, MA) were used for immunoblotting.

Techniques: Ligation, Injection, Western Blot, Expressing, Staining