Supplementary Material
Supplementary Methods
In vitro exposure to the inhibitors ofrespective pathways
MSCswere cultured overnight in 6-well plateswith DMEM containing 10% FBS for 24 hour, followed by being starved in 2% FBS for another 24 hoursprior to experimental interventions. The starved MSCs were then exposed to PI3K inhibitor (LY294002)or MAPK inhibitor (U0126) at the prescribed concentrations for 24 hours.The phosphorylations of Akt and ERK1/2 were detected by Western blotting.Three independent experiments were performed.
In vitro exposure to LDL
After a pre-incubation of 24 hrs, MSCs were cultured in different concentrations of LDL (density = 1.006-1.063g/mL; Merck, Germany)for another 24 hours as follows: 0, 20, 50, 100 and 200μg/mL. Atime-dependentexperiment was parallelly performed, in which MSCs were cultured with 100μg/mL of LDL at different intervals as follows: 0, 6, 12 and 24 hours, respectively. Afterwards, MTT assay was employed to measure the MSCs viability, and Western blotting, to examine the changes of p21 expression, Akt and ERK1/2 phosphorylations. Three independent experiments were performed for each experimental condition.
Supplementary Results
The effects of inhibitors on the activities of pathways
Western blotting showed that the effect of LY294002 onthe activity of PI3K/Akt pathway in MSCs was dose-dependent(Supplementary Figure 1a). The phosphorylation of Aktin MSCs, treated with LY294002 at a concentration of 10μmol/L, was reduced when compared with that in control group (0.72±0.07 vs. 1.00±0.18, p0.05), but decreased significantly at a rise ofLY294002 concentration [0.25±0.03 (25μmol/L), 0.13±0.04 (50μmol/L) vs.1.00±0.18 (control), p both< 0.05]. Similarly, the activity of MAPK/ERK1/2pathway in MSCs cultured with U0126 at a concentration of 5μmol/L exhibited a down-regulated trend compared with that of the control group (0.68±0.18vs.1.00±0.23, p0.05), and a significant decrease in comparison with that of the control group whenU0126 concentration rose to 10μg/mL or 20μg/mL (1.00±0.23vs.0.12±0.06, 0.10±0.05, pboth < 0.01)(Supplementary Figure 1b).
The effect of LDL on MSCs proliferation
MTT assay demonstrated that LDLwas not able to induceremarkablechanges in MSCs viabilities[Time-dependent: 0.82±0.13(6hr), 0.82±0.11(12hr),0.81±0.11 (24hr) vs.0.81±0.12 (control), pall > 0.05; Concentration-dependent:0.82±0.12(20μg/mL), 0.82±0.14(50μg/mL),0.81±0.11 (100μg/mL), 0.82±0.13 (200μg/mL)vs.0.81±0.12 (control), pall > 0.05](Supplementary Figure 2). Similarly, LDL presented no significant effect on the expression of p21 in MSCs[Time-dependent: 1.08±0.16 (6hr), 1.09±0.12 (12hr),1.15±0.19 (24hr) vs.1.00±0.17 (control), pall 0.05; Concentration-dependent:1.19±0.12 (20μg/mL), 1.14±0.15 (50μg/mL),1.15±0.19 (100μg/mL), 1.18±0.18 (200μg/mL) vs.1.00±0.17 (control), pall > 0.05](Supplementary Figure 3).
The effect of LDL on the activities of PI3K/Akt and MAPK/ERK1/2
Western blotting revealed that LDL exerted no statistical effect on the phosphorylation of Akt both in the time-dependent experiment and in the concentration-dependent one [Time-dependent: 1.16±0.15 (6hr), 1.13±0.14 (12hr),1.15±0.12 (24hr) vs.1.00±0.13 (control), pall > 0.05; Concentration-dependent:1.16±0.15 (20μg/mL), 1.17±0.14 (50μg/mL),1.15±0.12 (100μg/mL), 1.26±0.21 (200μg/mL) vs.1.00±0.13 (control), pall > 0.05](Supplementary Figure 3). The similar results could be found in the examination of the phosphorylation of ERK1/2 [Time-dependent: 1.13±0.13 (6hr), 1.15±0.15 (12hr),1.09±0.17 (24hr) vs.1.00±0.12 (control), pall > 0.05; Concentration-dependent:1.13±0.10 (20μg/mL), 1.19±0.15 (50μg/mL),1.09±0.17 (100μg/mL), 1.07±0.16 (200μg/mL) vs.1.00±0.12 (control),pall > 0.05].
Supplementary Figure legends
Supplementary Figure 1.Western blotting displayed that the inhibitors effectively down-regulated the activities of respective pathways in MSCs. (a) MSCs treated with or without LY294002at various concentrations for 24 hrs. (b) MSCs cultured with or withoutU0126at different concentrations for 24 hrs. *p＜0.05 vs. pAkt in control group; ** , ## p＜0.01 vs. pAkt, pERK in control group, respectively
Supplementary Figure 2.MTT assay demonstrated that LDLwas not able to induceremarkablechanges in MSCs proliferation. (a) Time-dependent: MSCs incubated with LDL under a concentration of 100μg/ml at the prescribed intervals; (b) Concentration-dependent: MSCs exposed to various concentrations of LDL for 24 hrs
Supplementary Figure 3.Western blottingshowed that LDLexerted not significant effect on p21 expression, the phosphorylations of Akt and ERK1/2. (a,b) MSCstreated with or without LDL (100μg/ml) for various periods, respectively; (c,d) MSCs incubated with or without LDL at various concentrations for 24 hrs
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