Journal: Scientific Reports

Article Title: Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

doi: 10.1038/srep25802

Figure Lengend Snippet: SaOs-2 cells were differentiated (Diff.) with ascorbic acid (AA) and β-glycerophosphate (βG) for 14 days before calcification was quantified by Alizarin Red staining followed by spectrophotometry. ( A ) AA and βG induced significantly more calcification relative to untreated, age-matched controls (***p < 0.001 t-test). ( B ) Intensity of Alizarin Red staining was greater in AA and βG treated cells relative to undifferentiated cells (Ctrl). ( C ) Calcitriol attenuated calcification in vitro in a dose dependent fashion (*p < 0.05; one way ANOVA) relative to differentiated (AA + βG) controls treated with vehicle (DMSO). Bars represent means of 3–4 independent experiments ± SEM.

Article Snippet: How to cite this article : Keasey, M. P. et al . Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

Techniques: Staining, Spectrophotometry, In Vitro

Journal: Scientific Reports

Article Title: Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

doi: 10.1038/srep25802

Figure Lengend Snippet: SLC20A2, SLC20A1 and XPR1 expression was assessed by RT-qPCR in AA/βG treated SaOs-2 cells co-incubated with calcitriol. ( A , B ) SLC20A2 mRNA expression was significantly higher in cells co-incubated with calcitriol at 7 ( A ) and 14 ( B ) days relative to AA/βG and vehicle treated controls. SLC20A1 ( C , D ) and XPR1 ( E , F ) were not affected it either time point. Bars represent means of 3 independent experiments ± SEM with one-way ANOVA (p < 0.05*, 0.01**).

Article Snippet: How to cite this article : Keasey, M. P. et al . Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

Techniques: Expressing, Quantitative RT-PCR, Incubation

Journal: Scientific Reports

Article Title: Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1

doi: 10.1038/srep25802

Figure Lengend Snippet: SaOs-2 cells were transfected with guide-RNA against SLC20A2 together with Cas-9. ( A ) Gene knockdown was confirmed by PCR amplification of the targeted region and T7 digests to confirm site cutting. Note the reduction in ~500 bp fragments and the appearance of shorter fragments in the CRISPR cells, which is the result of mismatch pairing of the DNA after repair by non-homologous end joining. ( B ) Primers were designed to flank the guide-RNA targeting site and genomic PCR performed on DNA from cells transfected with a donor plasmid (containing a Puromycin resistance gene flanked by homologous arms complimentary to the gene insertion site) with Cas-9. The reduced presence of a ~500 bp fragment is caused by the puromycin insertion, extending the size of the amplicon, thereby producing a diminished band in the PCR reaction. ( C ) Western blot analysis confirmed that protein expression was reduced in CRISPR-treated cells (representative of n = 2). ( D ) Wild type (WT) or SLC20A2 knock down (KD) SaOs-2 cells were treated for 14 days under calcifying conditions with or without calcitriol (Calc). KD cells were no longer protected from calcification by calcitriol (n = 3 independent experiments, *p < 0.05 by two-way ANOVA).

Article Snippet: How to cite this article : Keasey, M. P. et al . Vitamin-D receptor agonist calcitriol reduces calcification in vitro through selective upregulation of SLC20A2 but not SLC20A1 or XPR1.

Techniques: Transfection, Knockdown, Amplification, CRISPR, Non-Homologous End Joining, Plasmid Preparation, Western Blot, Expressing