Figure 1. Amino Acid Effects on BMSCs.
Panel A: (A) Effect of low amino acid levels on BMSC survival or of amino acid supplementation at a 1x (B) or 0.5x (C) level for three days followed by cell staining with Coomassie Blue. Higher magnification images are shown in the panels below. Essentially no BMSCs survived at the end of the three-day culture in M199 (A). Increasing concentrations of the amino acid mixture resulted in increased cell survival (B,C). Images representative of fifteen independent experiments. (D) Unstained cells grown in 1x amino-acid mixture.
Panel B: RT-PCR for amino acid sensors: Total RNA was extracted from BMSC cells with the RNeasy mini kit (Qiagen). cDNA was synthesized using 1 μg total RNA (SuperScript III First-Strand Synthesis System, Invitrogen) and PCR performed as described in Methods.
Panel C: RT-PCR for GPRC6A: To evaluate GPRC6A expression in bone cells we ran additional controls using the same conditions as in Panel B. As seen, we could detect GPRC6A in primary calvarial osteoblasts. Additional mouse tissues were used as positive and negative controls. OS= primary mouse calvarial osteoblasts; Hrt= mouse heart; Intest= mouse intestine; Kdn=mouse kidney, H20 was a negative control