Supplementary Material for: Amelioration of Uremic Toxin Indoxyl Sulfate-Induced Osteoblastic Calcification by SET Domain Containing Lysine Methyltransferase 7/9 Protein

<b><i>Background:</i></b> Vascular calcification (VC) is a very common phenomenon in patients with chronic kidney disease (CKD). It has been reported that some histone methylation play a role in VC as an epigenetic regulator. Indoxyl sulfate (IS) is a protein-bound uremic toxin that has been proven as one of the major risk factors of cardiovascular disease in CKD. SET domain containing lysine methyltransferase 7/9 (SET7/9) is one of the important histone methyltransferases. <b><i>Objectives:</i></b> This study aimed to determine the effect of IS on the expression of SET7/9 and the role of SET7/9 in IS-induced osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs). <b><i>Methods:</i></b> VSMCs were incubated with various concentrations of IS for different durations to assess osteoblastic differentiation and expression of SET7/9. Western blot analysis and quantitative real-time polymerase chain reaction were performed to assess the protein and mRNA levels of SET7/9 respectively. The calcium content was measured to evaluate calcification. <b><i>Results:</i></b> Osteoblastic differentiation and calcification of VSMCs and downregulation of the expression of SET7/9 were observed after IS treatment. The autophagy was activated after treatment with IS, whereas the inhibition of the autophagy partially attenuated the effect of IS on both the stimulation of the expression of runt-related transcription factor 2 and calcium deposition. <b><i>Conclusions:</i></b> Our data demonstrated that SET7/9 downregulation and autophagy activation may be the key mechanism of IS-induced VC in CKD.