Additional file 1 of Deciphering the effect of phytosterols on Alzheimer’s disease and Parkinson’s disease: the mediating role of lipid profiles

Supplementary Material 1

Del301-303 α_2BAR has a stronger interaction with spinophilin than WT α_2BAR.

<p>(A) Cells co-expressing Myc-spinophilin together with HA-tagged WT or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs). (B) Quantitation of the agonist-induced fold change of Myc-spinophilin in the IP complex with the WT α_2B or Del301-303 α_2BAR. n = 3–5 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303 α_2B.</p

Arrestin 3 and spinophilin reciprocally regulate agonist-induced α_2BAR phosphorylation.

<p>(A) CosM6 cells co-expressing HA-α_2BAR together with GFP-tagged arrestin 3 (GFP-Arr3) or GFP alone (vector) were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs) for indicated time points. Overexpression of GFP-Arr3 increased the phosphorylation level of α_2BAR following epinephrine stimulation. (B) Quantitation of agonist-induced fold change in α_2BAR phosphorylation. Data were mean ± SEM. n = 5 for each condition. ***, <i>p</i><0.001 by unpaired Student’s <i>t</i> test, GFP-Arr3 <i>vs</i>. vector control. (C) HEK293 cells co-expressing HA-α_2BAR with or without Myc-spinophilin were stimulated. Overexpression of Myc-spinophilin (Myc-Sp) reduced the phosphorylation level of α_2BAR following epinephrine stimulation. (D) Quantitation of agonist-induced fold change in α_2BAR phosphorylation. Data were mean ± SEM. n = 3 for each condition. **, <i>p</i><0.01, Myc-Sp <i>vs</i>. vector control.</p

The α_2BAR dependent hypertensive response is enhanced in arrestin 3 deficient mice.

<p>(A) Mean arterial pressure (MAP) measured in Arr3^-/-and corresponding Arr3^+/+ mice in the same genetic background after injection of UK14,304 (0.1mg/kg i.v.). (B) Quantitation of agonist-induced changes in MAP(ΔMAP) over the basal level. (C) Quantitation of area under curve of the hypertensive response curve. n = 5 for each group. *, <i>p</i> <0.05, Arr3^+/+<i>vs</i>. Arr3^-/-.</p

The α_2BAR dependent hypertensive response is diminished in spinophilin deficient mice.

<p>(A) Mean arterial pressure (MAP) measured in Sp^+/+ and Sp^-/- mice in the same genetic background after UK14,304 injection (0.1mg/kg i.v.). (B) Quantitation of agonist-induced changes in MAP(ΔMAP) over the basal level. (C) Quantitation of area under curve of the hypertensive response curve. n = 5 for each group. *, <i>p</i> <0.05, Sp^+/+<i>vs</i>. Sp^-/-.</p

Spinophilin is required for maintaining the sustained ERK1/2 activation induced by the Del301-303 α_2BAR in MEFs.

<p>(A) Sp^+/+ MEFs expressing the WT or Del301-303 α_2BAR were stimulated with 1μM clonidine for indicated time points. Representative blots show phospho- and total-ERK1/2. (B) Quantitation of ERK1/2 activation in Sp^+/+ MEFs. The relative ERK1/2 activation at each time point was expressed as a ratio to the peak level of ERK1/2 activation in the same experiment, which was arbitrarily defined as 1.0. n = 7 for the WT α_2B group and n = 6 for the Del301-303 α_2B group. *, <i>p</i><0.05; ***, <i>p</i><0.001, WT α_2B<i>vs</i>. Del301-303 α_2B. (C) Sp^-/- MEFs expressing the WT or Del301-303 α_2BAR were stimulated with 1μM clonidine for indicated time points. Representative blots show phospho- and total-ERK1/2. (D) Quantitation of ERK1/2 activation in Sp^-/- MEFs. n = 4 for the WT α_2B group and n = 3 for the Del301-303 α_2B group.</p

Spinophilin and arrestin reciprocally regulate α_2BAR-induced ERK1/2 activation kinetics in MEFs.

<p>(A) Arr2,3^-/- and corresponding WT (Arr2,3^+/+) MEFs expressing HA-α_2BAR were stimulated with 1μM clonidine for indicated time points. Phospho- and total-ERK1/2 were detected by Western blots. Representative blots from multiple independent experiments are shown. (B) Quantitation of ERK1/2 activation in Arr2,3^+/+ or Arr2,3^-/- MEFs at indicated time points. The relative ERK1/2 activation at each time point was expressed as a ratio to the peak level of ERK1/2 activation in the same experiment, which was arbitrarily defined as 1.0. Data were mean ± SEM. n = 4 for each condition. *, <i>p</i><0.05, Arr2,3^-/-<i>vs</i>. Arr2,3^+/+. (C) Sp^-/- and corresponding WT (Sp^+/+) MEFs expressing HA-α_2BAR were stimulated. Representative blots for phospho- and total ERK1/2 from multiple independent experiments are shown. (D) Quantitation of ERK1/2 activation in Sp^+/+ or Sp^-/- MEFs at indicated time points. Data were mean ± SEM. n = 7 for data collected in Sp^+/+ cells and n = 4 for Sp^-/- cells. *, <i>p</i><0.05; ***, <i>p</i><0.001, Sp^-/-<i>vs</i>. Sp^+/+.</p

Overexpression of spinophilin aa151-444 sufficiently attenuates α_2BAR phosphorylation.

<p>(A) HEK293 cells co-expressing HA-α_2BAR t.ogether with or without Myc-Sp151-444 were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs) for indicated time points. (B) Quantitation of agonist-induced fold change in α_2BAR phosphorylation. Data were mean ± SEM. n = 4 for each condition. **, <i>p</i><0.01; ***, <i>p</i><0.001 by unpaired Student <i>t</i> test, Myc-Sp151-444 <i>vs</i>. vector control. (C) Overexpression of Sp151-444 showed no effect on α_2BAR phosphorylation in CosM6 cells, which have a low level of endogenous arrestin expression. CosM6 cells co-expressing HA-α_2BAR together with or without Sp151-444 were stimulated. Representative blots from multiple independent experiments are shown.</p

The Del301-303 α_2BAR shows impaired interaction with arrestin 3.

<p>(A) Agonist treatment failed to promote interaction of the Del301-303 α_2BAR with arrestin 3. Cells co-expressing GFP-tagged arrestin3 (GFP-Arr3) with HA-tagged WT α_2BAR or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol to block βARs), and the interaction between arrestin and either WT or Del301-303 α_2BAR was examined by co-IP assays. (B) Quantitation of the agonist-induced fold change of GFP-Arr3 in the IP complex with the WT or Del301-303 α_2BAR. n = 3–4 for each condition. **, <i>p<</i>0.01, WT <i>vs</i>. Del301-303. (C) Del 301–303 α_2BAR was unable to interact with constitutively active mutant arrestin3 R170E following agonist stimulation. Cells co-expressing GFP-Arr3R170E together with WT or Del301-303 α_2BAR were stimulated with 100μM epinephrine (plus 1μM propranolol). (D) Quantitation of the agonist-induced fold change of GFP-Arr3R170E in the IP complex with the WT or Del301-303 α_2BAR. n = 3–4 for each condition. *, <i>p<</i>0.05, WT <i>vs</i>. Del301-303.</p

Effects of AST and ASIV on TNFα-induced up-regulation of ICAM-1 and E-selectin mRNAs.

<p>Confluent mAECs were pre-incubated with 250 µg/mL of AST or ASIV for 2 h, then washed and incubated for 6 h with 30 ng/mL TNFα. Cells were then collected and analyzed for ICAM-1 expression using real-time qPCR. Data are representative of three independent experiments with determinations made in triplicate and shown as mean values+S.D. **<i>P</i><0.01 compared with cells treated with TNFα alone. CN: Control.</p