primary antibodies against (vegf, tgfβ1, fgf2, igf‑1, scf, and il‑6)  (Santa Cruz Biotechnology)

Journal: Journal of Agricultural and Food Chemistry

Article Title: Sodium Benzoate Inhibits Osteoblast Differentiation and Accelerates Bone Loss by Regulating the FGF2/p38/RUNX2 Pathway

doi: 10.1021/acs.jafc.5c01124

Figure Lengend Snippet: NaB inhibits FGF2 expression in vitro and in vivo . (A) MC3T3-E1 cells and BMSCs were treated with or without 10 mM NaB. The cells were then subjected to RNA sequencing analysis, and a heatmap was generated to show differentially expressed genes (DEGs). Volcano plot of DEGs analyzed by RNA-seq of MC3T3-E1 cells (B) and BMSCs (C) treated with 0 or 10 mM NaB. FGF2 mRNA (D) and protein levels (E) in MC3T3-E1 cells treated with NaB (0, 5, or 10 mM) in OIM for 4 days. GAPDH was used as a loading control. (F) Quantification of the FGF2 protein level as shown in (E). (G) IF staining for FGF2 in MC3T3-E1 cells cultured in OIM for 4 days, scale bar = 50 μm. (H) IHC staining for FGF2 in the distal femur of mice, scale bar = 100 μm. Data are presented as the mean ± SD * P < 0.05; ** P < 0.01 compared with the 0 mM group.

Article Snippet: IHC was conducted using the primary antibody against FGF2 (1:100, ABclonal, Wuhan, China) and a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (H+L) secondary antibody (1:400, ABclonal, Wuhan, China).

Techniques: Expressing, In Vitro, In Vivo, RNA Sequencing, Generated, Control, Staining, Cell Culture, Immunohistochemistry

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2

doi: 10.3892/etm.2022.11433

Figure Lengend Snippet: miR-146a-5p directly targets FGF2 and FGF2 knockdown decreases collagen I and α-SMA expression levels in CFs. (A) StarBase database showed the potential binding sites between FGF2 and miR-146a-5p. (B) Luciferase reporter assay was used to determine the binding ability between FGF2 and miR-146a-5p in 293 cells. * P<0.05 vs. NC mimics. (C) RT-qPCR and (D) western blotting were used to assess the mRNA and protein levels of FGF2 in ISO-treated CFs transfected with NC or miR-146a-5p mimics or inhibitor or inhibitor. (E) RT-qPCR and (F) western blotting showed mRNA and protein levels of FGF2 in CFs transfected with siNC or siFGF2. (G) RT-qPCR analysis showed collagen I and α-SMA mRNA levels in ISO-treated CFs transfected with siNC or siFGF2. * P<0.05 vs. siNC. miR, microRNA; FGF2, fibroblast growth factor 2; α-SMA, α-smooth muscle actin; NC, negative control; CF, cardiac fibroblast; RT-q, reverse transcription-quantitative; ISO, isoproterenol; si, small interfering; WT, wild-type; MUT, mutant.

Article Snippet: After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibodies against FGF2 (1:1,000; cat. no. PB0619; Boster Biological Technology, Ltd.) and GADPH (1:1,000; cat. no. ab9485; Abcam) overnight at 4 ̊C.

Techniques: Knockdown, Expressing, Binding Assay, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Reverse Transcription, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2

doi: 10.3892/etm.2022.11433

Figure Lengend Snippet: miR-146a-5p suppresses ISO-treated cardiac fibrosis by targeting FGF2. (A) RT-qPCR and (B) western blotting were performed to determine the mRNA and protein levels of FGF2 in ISO-treated CFs transfected with NC or miR-146a-5p mimics or miR-146a-5p mimics + pcDNA3.1/FGF2. (C) RT-qPCR and (D) Cell Counting Kit-8 assay determined collagen I and α-SMA expression levels, as well as viability, in ISO-treated CFs transfected with NC or miR-146a-5p mimics or miR-146a-5p mimics + pcDNA3.1/FGF2. * P<0.05. miR, microRNA; ISO, isoproterenol; FGF2, fibroblast growth factor 2; RT-q, reverse transcription-quantitative; CF, cardiac fibroblast; NC, negative control; OD, optical density.

Article Snippet: After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibodies against FGF2 (1:1,000; cat. no. PB0619; Boster Biological Technology, Ltd.) and GADPH (1:1,000; cat. no. ab9485; Abcam) overnight at 4 ̊C.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Cell Counting, Expressing, Reverse Transcription, Negative Control

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: E2 and G-1 induce FGF2 expression through GPER in CAFs. 10 nM E2 ( a ) and 100 nM G-1 ( b ) induced FGF2 mRNA expression, as evaluated by quantitative PCR (qPCR). Values were normalized to 18S expression and shown as fold changes of FGF2 mRNA expression upon E2 and G-1 treatments respect to cells exposed to vehicle (). Each column represents the mean ± standard deviation (SD) of three independent experiments performed in triplicate. (**) indicates p < 0.01 and (*) indicates p < 0.05. ( c , d ) FGF2 protein expression by immunofluorescence in CAFs transfected for 24 h with control shRNA (panels 1–9) or sh G protein estrogen receptor (shGPER) (panels 10–18) and then treated for 6 h with vehicle, 10 nM E2 and 100 nM G-1, as indicated. FGF2 accumulation is shown by the green signal, nuclei are stained by 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue signal), scale bar = 100 μm. Images shown are representative of two independent experiments.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Immunofluorescence, Transfection, Control, shRNA, Staining

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: GPER mediates the up-regulation and the secretion of FGF2 by E2 and G-1 in CAFs. FGF2 protein expression by immunofluorescence in CAFs treated for 6 h with vehicle, 10 nM E2 and 100 nM G-1, alone (panels 1–9) ( a ) and in combination with 100 nM GPER antagonist G15 (panels 10–18) ( b ). FGF2 accumulation is shown by the green signal, nuclei are stained by DAPI (blue signal), scale bar = 100 μm. Images shown are representative of two independent experiments. ( c ) ELISA of FGF2 levels in supernatants collected from CAFs treated for 18 h with vehicle (-), 10 nM E2 and 100 nM G-1 alone and in combination with 100 nM GPER antagonist G15. Each column represents the mean ±SD of three independent experiments performed in triplicate. (**) indicates p < 0.01.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: The oncogene fos (c-fos) is involved in the up-regulation of FGF2 by E2 and G-1 in CAFs. 10 nM E2 ( a ) and 100 nM G-1 ( b ) induced c-fos mRNA expression, as evaluated by qPCR. Values were normalized to 18S expression and shown as fold changes of c-fos mRNA expression upon E2 and G-1 treatments respect to cells exposed to vehicle (-). ( c ) The treatment for 3 h with 10 nM E2 and 100 nM G-1 up-regulated c-fos protein, which is recruited to the AP-1 site located within the FGF2 promoter region (-1060/-848; the transcriptional start site is indicated as + 1), as ascertained by Chromatin Immunoprecipitation (ChIP)-qPCR assay ( d , e ). Data were normalized to the input and reported as fold changes respect to Immunoblobulin G (IgG). Each column represents the mean ±SD of three independent experiments performed in triplicate. In immunoblot experiments β-actin served as a loading control, side panels show densitometric analysis of the blot normalized to the loading control. (*) indicates p < 0.05 and (**) indicates p < 0.01. ( f ) FGF2 protein expression by immunofluorescence in CAFs transfected for 18 h with a vector (panels 1–9), or ( g ) with a construct encoding for a dominant negative form of c-fos (DN/c-fos) (panels 10–18) and then treated for 6 h with vehicle, 10 nM E2 and 100 nM G-1, as indicated. FGF2 accumulation is evidenced by the green signal, nuclei are stained by DAPI (blue signal), scale bar = 100 μm. Images shown are representative of two independent experiments.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Chromatin Immunoprecipitation, ChIP-qPCR, Western Blot, Control, Immunofluorescence, Transfection, Plasmid Preparation, Construct, Dominant Negative Mutation, Staining

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: Conditioned medium (CM) from estrogen-stimulated CAFs induces the activation of FGFR1- signaling pathway in MDA-MB-231 cells. ( a – c ) Phosphorylation of FGFR1, ERK1/2, AKT in MDA-MB-231 cells exposed for 1 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], alone and in the presence of 1 μM FGFR1 inhibitor PD173074. ( d , e ) Activation of ERK1/2 and AKT in FGFR1 (WT) MDA-MB-231 cells upon exposure for 1 h to CM from CAFs treated for 18 h with 10 nM E2 [CM/CAFs (+E2)], 100 nM G-1 [CM/CAFs (+G-1)]; ( f , g ) In FGFR1 (KO) MDA-MB-231 cells cultured in the same conditions as described above, the activation of ERK1/2 and AKT was no longer observed. FGF2 at 25 nM was used as positive control. FGFR1, ERK2, AKT and β-actin served as loading control, as indicated. Side panels show densitometric analysis of the blots normalized to the loading controls. Immunoblots shown are representative of three independent experiments. (*) indicates p < 0.05.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Activation Assay, Phospho-proteomics, Cell Culture, Positive Control, Control, Western Blot

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: Conditioned medium (CM) from estrogen-stimulated CAFs up-regulates CTGF levels through FGFR1 signaling pathway in MDA-MB-231 cells. ( a ) Pairwise linear regressions of FGFR1 versus CTGF mRNA levels were performed on METABRIC dataset of 1904 breast tumor samples. Scatter plot shows positive correlation between FGFR1 and CTGF expression. ( b – d ) CTGF mRNA and protein levels in FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control, evaluated by qPCR and western blot. In RNA experiments, values were normalized to the expression of 18S and shown as fold changes of CTGF mRNA expression upon CM from CAFs treated with E2 and G-1 respect to cells exposed to CM from CAFs treated with vehicle. Each column represents the mean ±SD of three independent experiments performed in triplicate. ( e – g ) Up-regulation of CTGF protein expression in MDA-MB-231 cells exposed for 3 h to CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)], or 100 nM G-1 [CM/CAFs (+G-1)] was no longer observed in the presence of 1 μM FGFR1 inhibitor PD173074, 10 μM MEK inhibitor PD98059 or 100 nM PI3K inhibitor Wortmannin (WM). β-actin served as loading control. Side panels show densitometric analysis of the blots normalized to the loading controls. Immunoblots shown are representative of three independent experiments. (**) indicates p < 0.01 and (*) indicates p < 0.05.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Positive Control, Western Blot, Control

Journal: Cells

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

doi: 10.3390/cells8030223

Figure Lengend Snippet: FGFR1 paracrine activation promotes migration and invasion in MDA-MB-231 cells. ( a ) FGFR1 (WT) and (b ) FGFR1 (KO) MDA-MB-231 cells were cultured for 8 h in CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control. Lines traced on cells were used to calculate the Polarity Index (PI). White lines define the migratory axis and black lines the transversal axis. PI = 1.0 indicates a polygonal shape, whereas a value > 1.0 defines ranges of migratory shapes. Scale bar = 30 μm. Images shown are representative of 30 random fields acquired in three independent experiments. Transwell assays were used to assess cell migration ( c ) and invasion ( d ) in FGFR1 (WT) and FGFR1 (KO) MDA-MB-231 cells cultured for 8 h in CM from CAFs treated for 18 h with vehicle [CM/CAFs (+vehicle)], 10 nM E2 [CM/CAFs (+E2)] or 100 nM G-1 [CM/CAFs (+G-1)], or exposed to 25 nM FGF2, as positive control. Cells were counted in at least 10 random fields at 10× magnification, in three independent experiments performed in triplicate. Scale bar = 200 μm, (**) indicates p < 0.01.

Article Snippet: Next, cells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, washed 3 times with PBS and incubated at 4 °C overnight with a mouse primary antibody against FGF2 (C-2) (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Activation Assay, Migration, Cell Culture, Positive Control