Serum and Urine Biochemistries and Gene Expression in <i>Fgfr1</i>^PT-cKO and <i>Fgfr1</i>^DT-cKO before and after rFGF-23 administration.

<p>Serum and Urine Biochemistries and Gene Expression in <i>Fgfr1</i>^PT-cKO and <i>Fgfr1</i>^DT-cKO before and after rFGF-23 administration.</p

Upregulation of calcium transport related genes in <i>Fgfr1</i>^PT-cKO and <i>Fgfr1</i>^DT-cKO mice.

<p>Gene expression in kidney of 16-weeks-old control, <i>Fgfr1</i>^PT-cKO_, and <i>Fgfr1</i>^DT-cKO mice was determined by real-time PCR as follows: Npt2a (A), Npt2c (B), Cyp27b1 (C), Cyp24a1 (D), TRPV5 (E), TRPV6 (F), CaBP28k (G), NCX1 (H), PMCA1b (I), and α-Klotho (J). Data represented mean ± S.D. of kidney samples (n = 5) from mice of each genotype. One-tail unpaired <i>t</i> test *<i>P<0</i>.<i>05</i>, **<i>P<0</i>.<i>01</i>, <i>***P<0</i>.<i>001</i> vs control.</p

Generation of FGFR1 conditional knockout in the distal tubule of mouse kidney.

<p>(A) Schematic deletion of FGFR1 gene in proximal tubule or distal tubule of kidney by crossing <i>FGFR1</i>^{<i>flox/flox</i>} female mouse with <i>γGT-Cre;Fgfr1</i>^{<i>null/+</i>} or <i>Ksp-Cre;Fgfr1</i>^{<i>null/+</i>} male mouse. (B) Genotyping of <i>Fgfr1</i>^PT-cKO mouse. (C) Genotyping of <i>Fgfr1</i>^{<i>DT-cKO</i>} mouse. (D) Expression of FGFR1 mRNA in the kidney of control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice as determined by real-time PCR. (E) Western blot analysis of expression of FGFR1 in the whole kidney of control, <i>Fgfr1</i>^{<i>PT-cKO</i>} or <i>Fgfr1</i>^DT-cKO mice. (F) Quantitation of FGFR1 Western blot. Expression of β-actin was used as internal controls. (G) Immunohistochemical staining of FGFR1 in the kidney of control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice. Letter G indicates glomeruli, and PT and DT indicate proximal and distal tubules, which have distinct morphological features. Magnification 400X. (H) Photo image of control and <i>Fgfr1</i>^PT-cKO mice. (I) Growth charts of <i>Fgfr1</i>^PT-cKO male and female (J). K. Photo image of control and <i>Fgfr1</i>^DT-cKO mice. (L) Growth charts of <i>Fgfr1</i>^DT-cKO male and female (M). Data were collected from mice (n = 5) of each genotype. One-tail unpaired <i>t</i> test *P<0.05 vs control.</p

Expression of Npt2a in <i>Fgfr1</i>^PT-cKO and <i>Fgfr1</i>^DT-cKO mice.

<p>Western blot analysis of expression of Npt2a from membrane proteins prepared from the kidney cortex of 16-weeks-old control, <i>Fgfr1</i>^PT-cKO, or <i>Fgfr1</i>^DT-cKO mice. (A) Western blot analysis of Npt2a expression. (B) Quantitation of Npt2a protein expression by Western blot analysis. (C) Immunohistochemical staining of Npt2a expression in kidney sections (arrow). The letter G indicates glomeruli. Protein samples isolated from pcDNA3.1 vector-transfected MDCK cells (ATCC) were used as negative control for Npt2a expression [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147845#pone.0147845.ref035" target="_blank">35</a>]. Protein samples isolated from MDCK cells transfected with Npt2a cDNA were used as positive controls. Data represent the mean ± S.D. of kidney samples derived from (n = 6) mice of each genotype. Images are representative of the analysis of five mice from each genotype. Magnification is 400X. One-tail unpaired <i>t</i> test *P<0.05 vs controls.</p

Conditional knockout of distal tubule FGFR1 results in elevated serum PTH.

<p>FGF-23 (A), calcium (B), phosphate (C), PTH (D), 1,25(OH)₂D (E), blood urea nitrogen (BUN) (F) were measured in serum samples collected from 16-weeks-old control, <i>Fgfr1</i>^PT-cKO, or <i>Fgfr1</i>^DT-cKO mice. Data were collected from mice (n = 5) of each genotype. One-tail unpaired <i>t</i> test *P<0.05 vs control.</p

Additional file 1 of Cirsiliol induces autophagy and mitochondrial apoptosis through the AKT/FOXO1 axis and influences methotrexate resistance in osteosarcoma

Additional file 1: Figure S1. A U2OS cells were treated with different concentrations of cirsiliol, stained with anti-Cyto-C and anti-TOMM20 antibodies and imaged using co-focused microscope imaging. Scale bars:100 µm. B Cyto-C and TOMM20 were quantified using FIJI/ImageJ. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Micro CT analysis of femur bone structure changes in control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice.

<p>Micro CT analysis of femur bone structure changes in control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice.</p

Conditional deletion of FGFR1 resulted in abnormal bone structures in <i>Fgfr1</i>^PT-cKO and <i>Fgfr1</i>^DT-cKO mice.

<p>(A) X-ray and μCT images of spinal bone of control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice. (B) μCT images of long bone from control, <i>Fgfr1</i>^PT-cKO, and <i>Fgfr1</i>^DT-cKO mice. Images are representative of samples collected from mice (n = 5) of each genotype.</p

<i>Fgfr1</i>^DT-cKO mice show urine calcium wasting and calcium deposition in the distal tubular of kidney.

<p>Urinary calcium (A), phosphate (B), volume (C), and creatinine (D) were assessed in a 12h urine collection in 16-weeks-old control, <i>Fgfr1</i>^{<i>PT-cKO</i>} or <i>Fgfr1</i>^DT-cKO mice. Kidney sections of control, <i>Fgfr1</i>^PT-cKO, or <i>Fgfr1</i>^DT-cKO mice. Kidney sections for H&E staining (E), magnification 200X and Von Kossa and Alizarin Red S staining (F), magnification 100X. Date were collected from mice (n = 5–6) of each genotype. One-tail unpaired <i>t</i> test *P<0.05 vs control.</p

Immunohistochemical staining of TRPV5 and α-Klotho.

<p>Kidney sections from 16-weeks-old control, <i>Fgfr1</i>^PT-cKO, or <i>Fgfr1</i>^DT-cKO mice were stained with TRPV5 or Klotho antibodies and counterstained with DAPI. Magnification of the images are 200X or 600X as indicated. Images data represented kidney of mice (n = 5) from each genotype.</p