Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques:

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 0, 7, 14, 21 and 28 days, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, **: p< 0,01 vs D0. Data are expressed as mean ± SD, n = 4. FGF23 production in media was quantified by ELISA (B), **: p< 0,01 vs D0, ***: p< 0,001, ND: Non Detectable. Total proteins were extracted from ATDC5 (0, 7, 14, 21 or 28 days post-insulin) and 10 μg was subjected to SDS-PAGE with FGF23 (1/200) antibody; ß-actin was used as loading control (C).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison, Enzyme-linked Immunosorbent Assay, SDS Page, Control

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 or 28 days and stimulated or not with 100ng/mL mouse rFGF23 for 24h, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance at D14 and D28 of FGF23 (A,B), COLX (C,D) and MMP13 (E,F) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p<0.05 vs ITS. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: ATDC5 were fixed with 4% PFA, and then stained by Alizarin red for 45min after 21 or 28 days of insulin stimulation with or without 100 ng/mL of mouse rFGF23. Images presented are representative of 5 independent experiments (A). ATDC5 micromasses were cultured for 14 days with insulin stimulation in the presence of 3 mM Pi or 100 ng/mL rFGF23 or both. Then they were fixed with 4% PFA and stained by Alizarin red for 45min. Images presented are representative of 3 independent experiments (B). Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 days, then reverse transcribed into cDNA and analysed by real-time PCR and compared to ITS. The relative abundance of COL X, MMP13 and FGF23 were normalized to RPS29 mRNA (C, D, E). Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to ITS. *: p < 0.05 vs ITS, #: p < 0.05 vs ITS + Pi and £: p < 0.05 vs ITS + rFGF23. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Staining, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 14).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Inhibition, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 28).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Inhibition, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 28 days pre-treated or not with 1 μM PD173074 one hour before stimulation with 100 ng/ml of rFGF23 (24h for RNA or 48h for proteins), then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A), MMP13 (B) and COL X (C) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p< 0.05 vs ITS, £: p< 0.01 vs rFGF23. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 3. Gal-1 (galectin-1) is induced by TNFα (tumor necrosis factor alpha) and enhances TNFα-induced MMP (matrix metalloprotease)-9 expression. A, Cultured vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were treated without (control [ctrl]) or with Ang II (angiotensin II; 100 nmol/L), TNFα (100 ng/mL), or IL (interleukin)-1β (10 ng/mL) as indicated for 48 h. Gal-1 expression in cell lysates was examined by Western blot analysis. B, The Gal-1 levels in culture media collected from treated cells described in A were measured by ELISA. Data shown are mean±SE of 3 independent experiments. *P<0.05 vs ctrl. C, Adventitial fibroblasts isolated from WT (wild type) and Gal-1−/− mice were treated with indicated concentrations of TNFα in culture for 48 h. Culture media were then collected and analyzed by gelatin zymography. Cell lysates were prepared and subjected to Western blot analysis using indicated antibodies. D, WT and Gal-1−/− fibroblasts were treated with TNFα (100 ng/mL) for indicated times. Cell lysates were prepared, and the expression levels of phosphorylated Erk1/2 and total Erk1/2 were examined by Western blot analysis. E, WT and Gal-1−/− fibroblasts were treated with or without TNFα (100 ng/mL) in the absence or presence of PD98095 (10 µmol/L) as indicated in culture for 48 h. Culture media and cell lysates were subjected to zymography and Western blot analysis, respectively.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Cell Culture, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Isolation, Zymography

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 4. Gal-1 (galectin-1) oxidation promotes MMP (matrix metalloprotease)-9 and inflammatory cytokine expression. A, Tissue lysates prepared from freshly isolated vessels of apoE-deficient mice were incubated with 2 mmol/L biotin-polyethylene oxide (PEO) iodoacetamide preceded with or without tris(2-carboxyethyl) phosphine hydrochloride (TCEP; 5 mmol/L) treatment at room temperature for 2 h in dark. The lysates were then subjected to immunoprecipitation with control IgG or anti-Gal-1 antibody as indicated. The levels of biotinylation and Gal-1 in cell lysates and immunoprecipitates were examined by streptavidin blotting and Western blot analysis, respectively. B, Cultured macrophages, vascular smooth muscle cells (VSMCs), and adventitial fibroblasts from Gal-1−/− mice were incubated with indicated concentrations of CSGal-1 (cysteine-less Gal-1 mutant) or oxidized Gal-1 (oxGal-1) for 24 (macrophages) or 48 h (VSMCs and fibroblasts). Culture media were harvested and analyzed by zymography. Cell lysates were prepared and subjected to Western blot analysis with MMP9 and β-actin antibodies. C, The levels of TNFα (tumor necrosis factor alpha), IL (interleukin)-6, and MCP-1 (monocyte chemoattractant protein-1) in culture media harvested from treated cells described in B were determined by ELISA. Data shown are mean±SE of 4 independent experiments. *P<0.05; **P<0.01, ***P<0.001 vs control.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Expressing, Isolation, Incubation, Immunoprecipitation, Control, Western Blot, Cell Culture, Mutagenesis, Zymography, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Gal-1 (Galectin-1) Upregulation Contributes to Abdominal Aortic Aneurysm Progression by Enhancing Vascular Inflammation.

doi: 10.1161/ATVBAHA.120.315398

Figure Lengend Snippet: Figure 5. MAP kinase pathways are implicated in oxidized Gal-1 (galectin-1; oxGal-1)–mediated induction of MMP (matrix metalloprotease)-9 and inflammatory cytokines. A, Cultured macrophages, vascular smooth muscle cells (VSMCs), and fibroblasts were treated with 1 µg/mL of oxGal-1 in culture for indicated times. The phosphorylation states of Erk, JNK, and p38 kinases were examined by Western blot analysis. B, Cultured macrophages, VSMCs, and fibroblasts were incubated without or with 1 µg/mL of oxGal-1 in the absence or presence of 10 µmol/L Erk inhibitor (PD98059), 10 µmol/L JNK inhibitor (SP600125), or 20 µmol/L p38 inhibitor (SB203580) as indicated for 24 (macrophages) or 48 h (VSMCs and fibroblasts). The cultured media were harvested, and the levels of indicated cytokines were determined by ELISA. Data shown are mean±SE of 3 to 4 independent experiments. Ctrl indicates control; IL-6, interleukin; MCP-1, monocyte chemoattractant protein-1; and TNFα, tumor necrosis factor alpha. *P<0.05; **P<0.01 vs oxGal-1–treated group without coincubation with respective kinase inhibitor.

Article Snippet: After collection and 3 washes, cells were resuspended in DMEM containing 10% FBS and 1 ng/mL of basic fibroblast growth factor (3139-FB; R&D Systems), followed by plating on 6-cm dish.

Techniques: Cell Culture, Phospho-proteomics, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet: GDF15 Expression Is Regulated by the Cellular ISR Pathway (A and C) GDF15 mRNA expression (A) and immunoblot analysis (C) of ISR components in wild-type (WT) mouse embryonic fibroblasts (MEFs) treated with vehicle control (Con), cobalt chloride (CoCl2, 625 μM), thapsigargin (Tg, 1 μM), tunicamycin (Tn 5 μg/mL), or L-Histidinol (His, 1 mM) for 6 h. (B) GDF15 mRNA expression in human cell lines (HeLA, HuH7, and A549) treated with Tn (5 μg/mL) for 6 h. (D) GDF15 mRNA expression in WT MEFs pre-treated for 1 h either with the PERK inhibitor GSK2606414 (GSK, 200 nM) or eIF2α inhibitor ISRIB (ISR, 100 nM), then co-treated with Tn (5 μg/mL) for a further 6 h. (E–G) GDF15 mRNA expression (E) in EIF2α Ser51 (SS) or phospho mutant (AA) MEFs or (F) in ATF4 wild-type (WT) or ATF4 knockout (KO) MEFs and (G) in control siRNA and CHOP siRNA transfected WT MEFs treated with Tn (5 μg/mL) for 6 h. (H) Diagram outlining pathway by which GDF15 and FGF21 expression is regulated by TN. mRNA expression is presented as fold expression relative to its respective control treatment for each cell type (set at 1) or TN-treated samples (set as 100) with normalization to HPRT gene expression in MEFs and GAPDH in human cells. Data are expressed as mean ± SD from at least three independent experiments. ∗∗∗ p < 0.001 versus control (con) for (A) and (B), and versus TN stimulated for (D)–(G) by two-tailed Student’s t test. Blots shown are representative of three independent experiments with Calnexin used as a loading control. See also Figures S3 and .

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Control, Mutagenesis, Knock-Out, Transfection, Gene Expression, Two Tailed Test

Journal: Cell Metabolism

Article Title: GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans

doi: 10.1016/j.cmet.2018.12.016

Figure Lengend Snippet:

Article Snippet: Mouse FGF21 was analyzed by FGF21 Quantakine ELISA (R&D Systems) following the manufacturer’s instructions.

Techniques: Recombinant, SYBR Green Assay, Protease Inhibitor, Reverse Transcription, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Control, TaqMan Assay, Software, Sterility, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Development (Cambridge, England)

Article Title: Evolutionarily conserved requirement of Cdx for post-occipital tissue emergence.

doi: 10.1242/dev.079848

Figure Lengend Snippet: Fig. 5. Addition of Fgf8 to the whole embryo culture medium of Cdx2 null embryos rescues their axial elongation. (A,B)E8.0 (early somite) embryos at the start of the culture (A, wild type; B, Cdx2 null). (C-F)Embryos after their culture for the same period without (C,D) or with (E,F) Fgf8 added to the culture medium. C and E are controls; D and F are Cdx2 null mutants. (G-J)Mox1 expression in another set of Cdx2 null mutants (H,J) and controls (G,I) that have been cultured for the same period with (I,J) and without (G,H) Fgf8. (K)Comparison of posterior elongation of Cdx2 null and control embryos cultured without and with Fgf8. y axis, total number of somites generated in culture; bars on the graph represent median values; n, number of embryos; several experimental data are superimposed as one symbol in the graph as they had the same value. (L)Statistical analysis of the axial growth rescue of Cdx mutants by Fgf8 using the Mann-Whitney U test. al, allantois; flb, forelimb bud. Anterior is to the right in A,B and up in C-J. Scale bars: 0.5 mm. See also supplementary material Fig. S3.

Article Snippet: Recombinant Fgf8 (isoform b) was purchased from R&D Systems (423- F8).

Techniques: Embryo Culture, Expressing, Cell Culture, Comparison, Control, Generated, MANN-WHITNEY