Journal: The FASEB Journal

Article Title: Lysophosphatidic Acid Synergizes With 1,25‐Dihydroxyvitamin D to Promote Fibroblast Growth Factor‐23 Synthesis via MAPK Signaling and Induction of the IL12A Gene

doi: 10.1096/fj.202502235R

Figure Lengend Snippet: Stimulation of FGF23 production by LPA and 1,25D and the whole transcriptome analysis of Ocy454 cells. (A) Secreted total FGF23 (cFGF23) concentrations in the culture medium of mouse long bone explants in response to vehicle (VEH), LPA alone, 1,25D alone, or LPA/1,25D after 24 h of stimulation. (B) Serum intact FGF23 (iFGF23) levels following the intraperitoneal LPA injection into the wild‐type and VDR KO littermates. (C) Experiment design for whole transcriptome analysis in Ocy454 cells. (D) Ven diagrams of differentially expressed genes (DEGs) in response to LPA, 1,25D, or LPA/1,25D treatment 2 and 8 h after stimulation. (E) Volcano plots of LPA and 1,25D responsive genes demonstrating LPA and 1,25D act on Ocy454 cells. (F) Expression profile of osteocyte genes in response to LPA, 1,25D, or the LPA/1,25D treatment after 2‐ and 8‐h stimulation. One‐way ANOVA followed by Tukey's (A) or two‐way ANOVA followed by Bonferroni (B) post hoc tests for multiple comparisons. Mean ± SEM, n = 3–4 experiments and n = 7–10 mice/group, respectively. DEGs and adjusted p values obtained by Benjamini‐Hochberg (BH) method using Limma package on R studio (D–F). Blue: 1,25D and Red: LPA responsive genes (E). Asterisks represent * p < 0.05, ** p < 0.01, *** p < 0.001 compared to vehicle‐treated group (F).

Article Snippet: Serum was collected 24 h later, and intact FGF23 (iFGF23) concentrations were measured using the iFGF23 (60–6800, Quidel) kit according to the manufacturer's instructions.

Techniques: Injection, Expressing

Journal: Scientific Reports

Article Title: Calcium and vitamin-D deficiency marginally impairs fracture healing but aggravates posttraumatic bone loss in osteoporotic mice

doi: 10.1038/s41598-017-07511-2

Figure Lengend Snippet: Serum analysis and gene expression and immunohistochemical analyses of the fracture calli of ovariectomized fractured mice fed a control (C), Ca/VitD-deficient (D) or Ca/VitD-supplemented (S) diet on day 23. ( a ) Relative alkaline phosphatase (ALP) gene expression in the fracture calli (n = 4–5/group). ( b ) Serum C-terminal telopeptide of type I collagen (CTX), and ( c ) serum N-terminal propeptide of type I procollagen (PINP) levels (n = 6–7/group). ( d ) Relative vitamin D-receptor (VDR) gene expression in the fracture calli (n = 4–5/group). ( e ) Serum fibroblast intact growth factor 23 (iFGF23) and ( f ) C-terminal FGF23 (cFGF23) levels (n = 5–7/group). ( g ) Ratio of serum iFGF23 to serum cFGF23 levels (n = 5/group). ( h ) Relative phosphate-regulating neutral endopeptidase, X-linked (Phex) gene expression in the fracture calli (n = 4–5/group). ( i ) Representative images of FGF23, fibroblast growth factor receptor 1 (FGFR1) and species-specific isotype control (rat and rabbit IgG) immunostained sections of the fracture calli. For gene expression analyses, β-2-microglobulin was used as the house-keeping gene. Data presented as the mean ± SD. Significant differences between C, D and S evaluated by ANOVA/Fishers LSD post-hoc : *p < 0.05, **p < 0.01.

Article Snippet: Paraffin-embedded sections of fractured femurs (d23) were immunohistochemically stained for FGF23 and fibroblast growth factor receptor 1 (FGFR1) using the following antibodies and dilutions: rat anti-mouse FGF23, 1:100 (MAB26291, R&D systems Inc., Minneapolis, USA), rabbit anti-mouse FGFR1/CD331, 1:50 (PA5-25979, Invitrogen, Thermo Fisher Scientific, Waltham, USA), goat anti-rabbit immunoglobulin G (IgG) (H + L) biotin-conjugated, 1:100 (B2770, Invitrogen), and goat anti-rat IgG (H + L) biotin-conjugated, 1:100 (A10517, Invitrogen).

Techniques: Gene Expression, Immunohistochemical staining, Control

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/elife.52709

Figure Lengend Snippet: Figure 6. Phytate-mediated Ca2+ deficiency promotes vitamin D insufficiency and renal phosphate wasting independent of FGF23 expression. (A–C) Time-course analysis of serum levels of intact PTH (A), 25(OH)D (B), and 1,25(OH)2D (C) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. For the early measurement of 25(OH)D (B) and 1,25(OH)2D, we pooled the sera from 2 to 3 rats. (D–F) Time-course analysis of renal CYP27B1 (D), CYP24A1 (E), and VDR (F) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (G) Immunoblot analysis of renal aKlotho, NHERF1, NaPi-2a in rats fed control, HP-LCa2+, Figure 6 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Rattus norvegicus) Sprague Dawley rat Orient Bio, Seoul, Korea Developed by Sprague Dawley, Inc. Chemical compound, drug AIN-93G Dyets Inc. DYET# 10700 Control diet Chemical compound, drug Phytic acid Sigma-Aldrich Cat. #: P-8810 Phytate diet Chemical compound, drug HNO3 Sigma-Aldrich Cat. #: 438073 Fecal mineral analysis Chemical compound, drug HF Sigma-Aldrich Cat. #: 695068 Fecal mineral analysis Chemical compound, drug HCl Sigma-Aldrich Cat. #: H1758 Fecal mineral analysis Chemical compound, drug EDTA Sigma-Aldrich Cat. #: 93283 Fecal phytate analysis Chemical compound, drug NaOH Sigma-Aldrich Cat. #: 415413 Fecal phytate analysis Chemical compound, drug Calcium Beckman Coulter OSR6113 Serum/urine biochemistry Chemical compound, drug Phosphate Beckman Coulter OSR6122 Serum/urine biochemistry Chemical compound, drug BUN (Urea) Beckman Coulter OSR6134 Serum/urine biochemistry Chemical compound, drug Creatinine Beckman Coulter OSR6178 Serum/urine biochemistry Chemical compound, drug Magnesium Beckman Coulter OSR6189 Serum/urine biochemistry Chemical compound, drug Urine protein Beckman Coulter OSR6170 Urine biochemistry Commercial assay or kit Rat intact PTH Immutopics Cat. #: 60–2500 ELISA kit Commercial assay or kit Human PTH Abcam Cat. #: ab230931 ELISA kit Commercial assay or kit Rat soluble RANKL Immundiagnostik Cat. #: K1019 ELISA kit Commercial assay or kit Rat osteoprotegerin Alpco Immunoassay Cat. #: 30–1020 ELISA kit Commercial assay or kit Rat intact FGF23 Elabscience Cat. #: E-EL-R3031 ELISA kit Commercial assay or kit Rat C-terminal FGF23 Elabscience Cat. #: E-EL-RB0377 ELISA kit Commercial assay or kit Human FGF23 Elabscience Cat. #: E-EL-H1116 ELISA kit Commercial assay or kit 25(OH)-Vitamin D Abbott/USA ARCHITECT 25-OH Vitamin D Chemiluminescence microparticle immunoassay Commercial assay or kit 1,25-(OH)two vitamin D DIAsoure 1,25(OH)2-VIT, D-RIA-CT/Belgium Radioimmunoassay Continued on next page Kim et al. eLife 2020;9:e52709.

Techniques: Expressing, Control, Western Blot

Journal: eLife

Article Title: High-phytate/low-calcium diet is a risk factor for crystal nephropathies, renal phosphate wasting, and bone loss

doi: 10.7554/elife.52709

Figure Lengend Snippet: Figure 6. Phytate-mediated Ca2+ deficiency promotes vitamin D insufficiency and renal phosphate wasting independent of FGF23 expression. (A–C) Time-course analysis of serum levels of intact PTH (A), 25(OH)D (B), and 1,25(OH)2D (C) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. For the early measurement of 25(OH)D (B) and 1,25(OH)2D, we pooled the sera from 2 to 3 rats. (D–F) Time-course analysis of renal CYP27B1 (D), CYP24A1 (E), and VDR (F) in rats fed control, HP-LCa2+, and HP-HCa2+ diets. (G) Immunoblot analysis of renal aKlotho, NHERF1, NaPi-2a in rats fed control, HP-LCa2+, Figure 6 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Genetic reagent (Rattus norvegicus) Sprague Dawley rat Orient Bio, Seoul, Korea Developed by Sprague Dawley, Inc. Chemical compound, drug AIN-93G Dyets Inc. DYET# 10700 Control diet Chemical compound, drug Phytic acid Sigma-Aldrich Cat. #: P-8810 Phytate diet Chemical compound, drug HNO3 Sigma-Aldrich Cat. #: 438073 Fecal mineral analysis Chemical compound, drug HF Sigma-Aldrich Cat. #: 695068 Fecal mineral analysis Chemical compound, drug HCl Sigma-Aldrich Cat. #: H1758 Fecal mineral analysis Chemical compound, drug EDTA Sigma-Aldrich Cat. #: 93283 Fecal phytate analysis Chemical compound, drug NaOH Sigma-Aldrich Cat. #: 415413 Fecal phytate analysis Chemical compound, drug Calcium Beckman Coulter OSR6113 Serum/urine biochemistry Chemical compound, drug Phosphate Beckman Coulter OSR6122 Serum/urine biochemistry Chemical compound, drug BUN (Urea) Beckman Coulter OSR6134 Serum/urine biochemistry Chemical compound, drug Creatinine Beckman Coulter OSR6178 Serum/urine biochemistry Chemical compound, drug Magnesium Beckman Coulter OSR6189 Serum/urine biochemistry Chemical compound, drug Urine protein Beckman Coulter OSR6170 Urine biochemistry Commercial assay or kit Rat intact PTH Immutopics Cat. #: 60–2500 ELISA kit Commercial assay or kit Human PTH Abcam Cat. #: ab230931 ELISA kit Commercial assay or kit Rat soluble RANKL Immundiagnostik Cat. #: K1019 ELISA kit Commercial assay or kit Rat osteoprotegerin Alpco Immunoassay Cat. #: 30–1020 ELISA kit Commercial assay or kit Rat intact FGF23 Elabscience Cat. #: E-EL-R3031 ELISA kit Commercial assay or kit Rat C-terminal FGF23 Elabscience Cat. #: E-EL-RB0377 ELISA kit Commercial assay or kit Human FGF23 Elabscience Cat. #: E-EL-H1116 ELISA kit Commercial assay or kit 25(OH)-Vitamin D Abbott/USA ARCHITECT 25-OH Vitamin D Chemiluminescence microparticle immunoassay Commercial assay or kit 1,25-(OH)two vitamin D DIAsoure 1,25(OH)2-VIT, D-RIA-CT/Belgium Radioimmunoassay Continued on next page Kim et al. eLife 2020;9:e52709.

Techniques: Expressing, Control, Western Blot

Journal: PLoS ONE

Article Title: Osteocyte-Specific Deletion of Fgfr1 Suppresses FGF23

doi: 10.1371/journal.pone.0104154

Figure Lengend Snippet: (A) Gross appearance, tail length, and body weight. Compared with control mice, Fgfr1 Dmp1-cKO -null mice had normal gross appearance and body weight. However, Hyp mice showed considerably shorter tail length and lower body weight, compound Hyp ; Fgfr1 Dmp1-cKO -null mice displayed intermediate tail length and body weight between control and Hyp mice. Data are mean ± S.D. from 5–6 individual mice. (B and C) Western blot analysis of total Fgf2 and Fgf23 protein levels in bone. A representative Fgf2, Fgf23, and β-Actin gel were shown in upper, middle, and lower panels of B, respectively. The intensity of bands was quantified using Image J software ( http://rsb.info.nih.gov/ij/ ), and the data shown in C are mean ± S.D. from three independent experiments. Values sharing the same superscript in different groups are not significantly different at P <0.05.

Article Snippet: Antibody against Fgf23 (MAB2629) was obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Control, Western Blot, Software

Journal: PLoS ONE

Article Title: Osteocyte-Specific Deletion of Fgfr1 Suppresses FGF23

doi: 10.1371/journal.pone.0104154

Figure Lengend Snippet: Gene-expression profiles in bone in 6-week-old mice.

Article Snippet: Antibody against Fgf23 (MAB2629) was obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques:

Journal: PLoS ONE

Article Title: Osteocyte-Specific Deletion of Fgfr1 Suppresses FGF23

doi: 10.1371/journal.pone.0104154

Figure Lengend Snippet: Biochemistry analysis in 6-week-old mice.

Article Snippet: Antibody against Fgf23 (MAB2629) was obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques:

Journal: PLoS ONE

Article Title: Osteocyte-Specific Deletion of Fgfr1 Suppresses FGF23

doi: 10.1371/journal.pone.0104154

Figure Lengend Snippet: MC3T3-E1 cells were co-transfected with various plasmids and treated with different drugs as described in Material and Methods. (A) Dose-dependent stimulation of mouse FGF23 promoter activity by recombinant FGF2 (5∼100 ng/ml); (B) Dominate-negative FGFR1(TK−) constructs blocked the stimulatory effect of recombinant FGF2 (50 ng/ml) on mouse FGF23 promoter activity; (C) Effect of PLCγ inhibitor (U73122) on FGF2-induced FGF23 promoter activity; (D) Effect of PI3K inhibitor Wortmannin (Wort) on FGF2-induced FGF23 promoter activity; (E) Dose-dependent inhibition of FGF2-induced FGF23 promoter activation by MAPK inhibitors (U0126). (F) Overexpression of HMW- Fgf2 constructs stimulated mouse FGF23 promoter activity. Data are expressed as the mean ± S.D. from triple three independent experiments. Values sharing the same superscript in different groups are not significantly different at P <0.05. * indicates significant difference from control vector group.

Article Snippet: Antibody against Fgf23 (MAB2629) was obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Transfection, Activity Assay, Recombinant, Construct, Inhibition, Activation Assay, Over Expression, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Osteocyte-Specific Deletion of Fgfr1 Suppresses FGF23

doi: 10.1371/journal.pone.0104154

Figure Lengend Snippet: MC3T3-E1 cells were co-transfected with pcDNA3.1- FGFR1 expression plasmids and human pcDNA3.1-FGF23-V5-His expression plasmids and treated with different drugs as described in Material and Methods. (A and B) Time-dependent stimulation of human FGF23-V5 protein expression by recombinant FGF2 (50 ng/ml); (C and D) Effect of PI3K inhibitor Wortmannin (Wort) and protein synthesis inhibitor Cycloheximide (CXM) on FGF2-induced human FGF23-V5 protein expression. Both Wortmannin and Cycloheximide completely blocked FGF2-induced FGF23 protein increase. Data are expressed as the mean ± S.D. from triple three independent experiments. Values sharing the same superscript in different groups are not significantly different at P <0.05.

Article Snippet: Antibody against Fgf23 (MAB2629) was obtained from R&D Systems, Inc. (Minneapolis, MN).

Techniques: Transfection, Expressing, Recombinant

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: Fgf23 expression is down-regulated by pTxA2 in UMR-106 and MC3T3-E1 cells. Arithmetic means ± SEM of rel. Fgf23 gene expression in UMR-106 ( a , n = 5) and MC3T3-E1 ( b , n = 5) cells treated without or with pTxA2 for 24 h ( a ) or 6 h ( b ). * p < 0.05. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Expressing, Gene Expression

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: Fgf23 expression is decreased by TxA2 agonist I-BOP in UMR-106 and MC3T3-E1 cells. Arithmetic means ± SEM of rel. Fgf23 gene expression in UMR-106 ( a , n = 6) and in MC3T3-E1 ( b , n = 7) cells treated without or with TxA2 agonist I-BOP for 24 h ( a ) or 6 h ( b ). * p < 0.05. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Expressing, Gene Expression

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: Fgf23 expression is inhibited by TxA2 agonist U46619 in UMR-106 and MC3T3-E1 cells. Arithmetic means ± SEM ( n = 5) of rel. Fgf23 gene expression in UMR-106 ( a ) and MC3T3-E1 ( b ) cells treated without or with TxA2 agonist U46619 for 24 h ( a ) or 6 h ( b ). * p < 0.05. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Expressing, Gene Expression

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: C-terminal FGF23 concentration in the supernatant of UMR-106 cells is lowered by TxA2 agonists. Arithmetic means ± SEM of C-terminal FGF23 concentration in the supernatant of UMR-106 cells treated without or with 100 ng/mL pTxA2 ( a , n = 7), 100 ng/mL I-BOP ( b , n = 7) or 5 µ m U46619 ( c , n = 8) for 24 h. * p < 0.05, ** p < 0.01. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Concentration Assay

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: TxA2 antagonist SQ29548 reverses the effect of I-BOP on Fgf23 mRNA transcripts. Arithmetic means ± SEM ( n = 10) of relative Fgf23 gene expression in UMR-106 cells treated without or with 100 ng/mL TxA2 agonist I-BOP in the presence or absence of 1 µ m TxA2 antagonist SQ29548 for 24 h. ** p < 0.01. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Gene Expression

Journal: Kidney & Blood Pressure Research

Article Title: Thromboxane A2 or Activated Platelets Slightly Lower Fgf23 Expression in vitro

doi: 10.1159/000545696

Figure Lengend Snippet: Exposure to activated platelets suppresses Fgf23 mRNA transcripts in UMR-106 cells. Arithmetic means ± SEM ( n = 8) of relative Fgf23 gene expression in UMR-106 cells exposed or not exposed to 100,000/µL freshly isolated human platelets in the presence or absence of 1 U/mL thrombin. * p < 0.05. SEM, standard error of the mean.

Article Snippet: The FGF23 protein concentration was determined using a mouse/rat C-terminal and a mouse/rat intact FGF23 ELISA kit (both from Quidel, San Clemente, CA, USA).

Techniques: Gene Expression, Isolation