1. Supplemental figure 1 ab CtrlPi 10 20 30 40 50 * %Cells with GFP-LC3 puncta  -actin LC3I LC3II Ctrl Pi (3 mM) CtrlPi 0.5 1.0 1.5 2.0 2.5 * LC3-II protein level Supplemental figure 1 Pi induced autophagy in T/G human aortic VSMCs (HA-VSMCs). T/G HA-VSMCs were transfected with GFP-LC3 plasmids and treated with Pi (3 mM) for 12h. (a) GFP signal was detected by confocal microscopy, and the proportion of cells with GFP-LC3 puncta was shown. (b) Western blot analyzed LC3 protein level. LC3-II density was standardized by  -actin. n=3, * P<0.05.

2. Supplemental figure 2 Anti-LC3 Von kossa staining Supplemental figure 2 LC3 puncta cells were found in both calcified and non-calcified aortic wall in chronic renal failure rats. Red arrows indicated autophagic cells, and red circle indicated calcified area.

3. Supplemental figure 3 Control ScrambleAtg5 siRNA 0.5 1.0 1.5 Relative Atg5 mRNA level * Supplemental figure 3 Knockdown of Atg5 by siRNA. Rat VSMCs were transfected with scramble or Atg5 small interfering RNA (siRNA) for 48 h. Quantitative real- time PCR analysis of Atg5 mRNA level. n=3, * P<0.05.

4. Supplemental figure 4 Control Pi Mn+Pi Mn+3-MA+Pi 3-MA+Pi Supplemental figure 4 TUNEL staining (red) to identify apoptotic cells. Nuclei were counterstained with Hochest (blue). BASMCs were treated with 3-MA (5 mM), MnTMPyP (Mn, 25  M), and/or Pi (3 mM) as indicated for 7 days.

5. Supplemental figure 5 a ControlPi3-MA3-MA+Pi 10 20 30 40 50 * * ALP activity (U/ g prot) b Supplemental figure 5 The effects of autophagy inhibition on release of alkaline phosphatase (ALP) into the matrix vesicle (MV) fraction. (a) ALP activity of BASMCs after treatment with Pi (3 mM), 3-MA (5 mM) or Pi+3-MA for 3 days. (b) Data are expressed as the ratio of the cumulative release of MV ALP/total cellular ALP to indicate the effects of the drug on the release of ALP into the MV fraction. * P<0.05 vs control, # P<0.05 vs Pi. 2 4 6 8 * * * # MV ALP/Cell ALP ControlPi3-MA3-MA+Pi

6. Actin a 0.2 0.4 0.6 0.8 1.0 SM-  -actin 0.5 1.0 1.5 Cbf  1 0.5 1.0 1.5 Msx2 Relative mRNA level CtrlPiCtrlPi 3-MA CtrlPiCtrlPi 0.5 1.0 1.5 3-MA SM22  * * Relative mRNA level CtrlPiCtrlPi 3-MA Relative mRNA level CtrlPiCtrlPi 3-MA Supplemental figure 6 * * Supplemental figure 6 The effects of autophagy inhibition on VSMC phenotypic transition. (a) Quantification of relative mRNA level of VSMC markers (SM22  and SM-  -actin) and osteogenic-related markers (core- binding factor  1 [Cbf  1/Runx2] and msh homeobox 2 [Msx2]) in BASMCs treated with Pi (3 mM), 3-MA (5 mM) or Pi+3-MA for 3 days. n=4, * P<0.05.

7. Control Pi 3-MA Pi+3-MA SM-  - actin b Supplemental figure 6 continued Supplemental figure 6 The effects of autophagy inhibition on VSMC phenotypic trasition. (b) Immunofluorescence distribution of actin and smooth muscle  -actin (SM-  -actin) in BASMCs treated with Pi (3 mM), 3- MA (5 mM) or Pi+3-MA for 3 days. Red arrows showed the polarized distribution of actin in the perinuclear region in 3-MA treated VSMCs. White arrows showed the microvillar processes at the cellular edges that appeared to be devoid or greatly depleted of actin staining. Actin