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2.5. *. 2.0. 1.5. LC3-II protein level. 1.0. 0.5. Ctrl. Pi. Supplemental figure 1. a. b. Ctrl Pi (3 mM). LC3I. LC3II. 50. -actin. *. 40. 30. %Cells with GFP-LC3 puncta. 20. 10. Ctrl. Pi.
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2.5 * 2.0 1.5 LC3-II protein level 1.0 0.5 Ctrl Pi Supplemental figure 1 a b Ctrl Pi (3 mM) LC3I LC3II 50 -actin * 40 30 %Cells with GFP-LC3 puncta 20 10 Ctrl Pi 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.
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.
Supplemental figure 3 1.5 1.0 Relative Atg5 mRNA level Supplemental figure 3Knockdown of Atg5 by siRNA. RatVSMCs 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. 0.5 * Control Scramble Atg5 siRNA
Supplemental figure 4 Control Mn+Pi Mn+3-MA+Pi 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.
Supplemental figure 5 a b 8 50 # * * * * 40 6 * 30 MV ALP/Cell ALP ALP activity (U/ g prot) 4 20 2 10 Control Pi 3-MA 3-MA+Pi Control Pi 3-MA 3-MA+Pi 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.
SM--actin Cbf 1 Msx2 1.0 1.5 1.5 0.8 1.0 1.0 0.6 0.4 0.5 0.5 0.2 Supplemental figure 6 a SM22 * * 1.5 * * 1.0 Relative mRNA level Relative mRNA level Relative mRNA level Relative mRNA level 0.5 Ctrl Pi Ctrl Pi Ctrl Pi Ctrl Pi Ctrl Pi Ctrl Pi Ctrl Pi Ctrl Pi 3-MA 3-MA 3-MA Actin 3-MA 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 [Cbf1/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.
Supplemental figure 6 continued Actin b Control Pi 3-MA Pi+3-MA SM--actin 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.
