Additional file 1: of Targeted lipidomics analysis identified altered serum lipid profiles in patients with polymyositis and dermatomyositis

Table S1. Lipid profiles in serum from patients with PM/DM and healthy individuals. Table S2. Lipid profiles in serum from patients with PM or DM before and after immunosuppressive treatment. (DOCX 44 kb

Maximum of two 5LO can bind to one ND.

<p>Calcium native PAGE analysis of ND and 5LO incubations in presence of Ca²⁺, with stoichiometries ranging from 4:1 to 0.25:1 (ND:5LO). From lanes 3 to 5, the concentration of ND is decreasing with constant 5LO. Lane 6, 5LO and ND are in equimolar ratio (0.22 μM). From lanes 7 to 9, the concentration of 5LO is increasing with constant ND. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152116#sec011" target="_blank">results</a> for further details.</p

The dimeric 5LO does not bind to nanodiscs or separate into monomeric 5LO.

<p>Native PAGE analysis of purified ND (Lane 1) and purified dimer 5LO (Lane 2). Dimeric 5LO incubated with ND with Ca²⁺ (Lane 3) shows only bands corresponding to the dimer (158 kDa) and the ND (260 kDa). Dimer 5LO incubated with ND but without Ca²⁺ (Lane 4) shows the same bands as for lane 3 indicating that the dimer is not split into monomers by the presence of a membrane.</p

Purified 5LO dimers shown by negative stain TEM.

<p>(A), Negative stain (uranyl formate 1%) image in which four boxed dimers are shown magnified in the gallery to the right. 100 nm and 10 nm scale bars, respectively. (B), Class averages showing dimers of 5LO that conform to the proposed dimer in <b>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152116#pone.0152116.ref013" target="_blank">13</a>]</b>. The box side length is 27.6 nm.</p

5LO can bind to nanodiscs in the presence of Ca²⁺.

<p>(A) Blue native PAGE (4–16% Bis Tris gel) analysis of ND-5LO complexes formed after incubations of ND and 5LO with and without 1 mM Ca²⁺ present. Lane 1, purified ND. Lane 2, purified 5LO. Lane 3, sample from incubation of 5LO, ND and Ca²⁺. Each lane is loaded with 1.5 μg of protein. The bands at ≈340 kDa (*) and ≈260 kDa (¤) were excised and subjected to SDS-PAGE. Lane 4, sample from incubation of 5LO and ND without Ca²⁺. (B) SDS PAGE (12% Tris-glycine gel) denaturing analysis of the two bands excised from BN-PAGE lane 3. Both excised bands contain MSP1E3D1 (32 kDa) but only the ≈340 kDa (*) band contain 5LO (78 kDa).</p

Recombinant 5LO exist as monomer as well as dimer.

<p>(A), Size exclusion chromatography of 5LO. Arrows indicate elution of standard proteins: Aldolase (158 kDa), Conalbumin (75 kDa). Peak 1 (retention volume 66 ml) corresponds to 5LO dimer. Peak 2 (retention volume 78 ml) corresponds to 5LO monomer. (B and C) SDS-PAGE of material in SEC peaks (lanes 1, from peak1; lanes 2, from peak 2) (B) Coomassie stained gel. (C) Western blot with 5LO antibody.</p

Both indirect and direct visualization of the effect of calcium on interaction of monomeric 5LO with ND, by negative stain electron microscopy.

<p>(A), ND showing stack formation induced by PTA stain, homogeneous size and the rigid structure of ND. (B), the ND preparation as in A but in the presence of Ca²⁺ shows the same amount of stacking as in A. (C), PTA-stained samples of equimolar concentrations of ND incubated with 5LO in calcium-free buffer, clearly showing stack formation, which signifies lack of interaction between 5LO and ND. (D), PTA stained samples of equimolar concentrations (0.8 μM) of ND incubated with 5LO in the presence of 1 mM Ca²⁺, showing very few stacks. A lack of PTA-induced stacking formation, signifies the interaction between 5LO and ND. The magnifications on the right in are representatives of the majority of the particles found in the corresponding image. (E), Class averages of 5LO-ND-complexes stained with PTA. Five images were used (including the one in D) to box 377 particles. Box-size is 20.8 nm. Scale bars are 10 nm and 100 nm respectively. (F), PTA stained samples of ND (0.8 μM) incubated with 5LO (1.6 μM) in the presence of 1 mM Ca²⁺, showing even fewer stacks. Most particles are larger and have different shapes compared to the 1:1 ratio shown in (D). (G), Class averages of 5LO-ND-complexes stained with PTA. Thirty six images were used to box 573 particles. Box-size is 26.6 nm. Scale bars are 10 nm and 100 nm respectively.</p

5LO dimers cannot bind on nanodiscs as shown by the extensive stacking after staining by PTA.

<p>(A) dimeric 5LO and ND incubated with Ca²⁺. Note, this preparation of nanodiscs was heterogenous and varied in the outer diameter of the discs, hence branches form (see boxes to the right). Circles show objects interpreted as unbound dimeric 5LO particles. (B) dimeric 5LO and ND incubated without Ca²⁺. Here, the preparation of nanodiscs did not vary in the outer diameter of the discs, hence no branches form. Instead, long stacks may lie on top of each other (top box) or lie parallel (middle and lower boxes). Dimeric 5LO are present (circles).</p

5LO initial velocity and product formation.

<p>Initial velocities (column V_init) and total 5-HPETE/5-HETE formation (column Final Product) were determined by UV spectroscopy at 235 nm, during 5 min incubations with different activating factors. Ratio 5LO:ND is 1.3:1. Data are mean±SEM (n = 3). LTA₄ formation (column LT) in 10 min incubations with different activating factors was measured by LC-MS/MS. * Not detected. Data are mean±SEM(n = 2).</p

Prostanoid profiles for WT MPGES1 and variants Asp49Ala, Arg73Ala, Arg73Leu, Arg126Ala, Arg126Leu, Ser127Ala, expressed in <i>E</i>. <i>Coli</i>.

<p>Prostanoid production was measured by LC-MS/MS after 60 seconds incubations of membrane fractions with PGH₂ (10μM final concentration) and GSH (2.5mM final concentration) at room temperature. Denatured enzymes by boiling were incorporated in the activity assay as controls. All prostanoid measurements of membrane fractions are expressed as mean ± SD from two independent experiment performed in duplicates.</p