Figure 2
Effects of low Pi loading on type-III NaPiT mRNA expression. cDNA reverse transcribed from the mRNA of SH-SY5Y or A172 cells was used as a template for the RT-PCR and qRT-PCR assays. (A) Expression of NaPiT mRNA in SH-SY5Y or A172 cells. For each amplification reaction, positive control reactions (PC) were performed using cDNA templates from the kidney (except for SLC34A2) or lung (for SLC34A2). Negative control reactions lacking RT reactions (RT-) were also performed to exclude the possibility of genomic DNA contamination. The sequences of primer sets are shown in Table 1. (B) The TaqMan-based qPCR assay using SLC20A1 and SLC20A2 probes. Data were normalized to the amount of 18s ribosomal RNA (18s rRNA), and results are expressed as the fold increase compared with that in the SH-SY5Y cells (mean ± SEM; n = 3). (C) Effect of PFA on cytotoxicity. (D,E) At 24 h after low Pi loading treatment, mRNA expressions of SLC20A1 and SLC20A2 was analyzed using the TaqMan-based qPCR assay. (F,G) SH-SY5Y and A172 cells were transiently transfected with SLC20A1 or SLC20A2 siRNA. At 24 h after transfection, mRNA expressions of SLC20A1 and SLC20A2 were analyzed using the TaqMan-based qPCR assay. (H,I) The Pi uptake assay was carried out in SLC20A1- and SLC20A2-suppressed cells. Data were normalized to the amount of 18s rRNA, and results are expressed as the fold increase compared with that at 1 mM Pi (D,E) or in NC (F–I) (mean ± SEM; n = 3). The significance of any difference was determined using ANOVA followed by the Bonferroni/Dunn post-hoc test (*p < 0.05).
