Additional file 4 of Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

Additional file 4: Supplemental Fig. 4. (A) Size distribution of APP Tg serum-derived EVs analyzed by a nanoparticle analyzer, qNano. (B) Representative image of APP Tg serum-derived EVs by electron microscopy. (C) Western blot analysis of Aß, ganglioside GM1, CD9, and ßIII tubulin in APP Tg serum-derived EVs. (D) Representative images of hippocampal sections immunostained with Aβ. Scale bars, 200 μm. (E) The levels of Aβ1-40 and Aβ1-42 in whole sera and serum-derived EVs of 12-month-old APP Tg mice were measured by conventional ELISA (n = 4 each). n.d., not detected

Suppression of the growth defects in flippase mutants by decreased synthesis of PI.

<p>(A) Effects of inositol depletion on steady state phospholipid composition in the flippase mutants. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted). Phospholipids were extracted and quantified by liquid chromatography mass spectrometry as described in the Materials and Methods. The data represent mole percentage (mol%) of total phospholipids, with mean values ± standard deviation (n = 3). Asterisks indicate a significant difference in the Student’s <i>t</i> test (*: <i>P</i> < 0.05; **: <i>P</i> < 0.005). The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (B) Suppression of the cold-sensitive growth defects in the <i>cdc50</i>Δ mutant by partial depletion of Pis1p. Cells were pregrown to early log phase in SD medium containing 2% raffinose and 2% or 0.005% galactose for 2 days, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto the same medium and YPDA, followed by incubation at the indicated temperature for the indicated time. The strains used were YKT1066 (wild type, WT), YKT1697 (<i>cdc50</i>Δ), YKT1942 (<i>P</i>_<i>GAL1</i><i>-PIS1</i>), and YKT1943 (<i>P</i>_<i>GAL1</i><i>-PIS1 cdc50</i>Δ). (C) Effects of Pis1p depletion on steady state phospholipid composition in the <i>cdc50</i>Δ mutant. Cells were pregrown and grown in SD medium containing 2% raffinose and 0.005% galactose for 4 days, in total, at 30°C. Phospholipids were extracted and quantified as in (A). The strains used were those in (B). (D) Aureobasidin A (AbA) treatment does not suppress the growth defects of flippase mutants. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. Cells were spotted onto a YPGA, YPDA, or YPDA containing 75 ng/ml AbA agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>). (E) <i>ART5</i> overexpression suppressed the growth defects of flippase mutants more efficiently than the <i>CHO1</i> overexpression. Cells were pregrown to early log phase in SG-Leu medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SDA agar plate with or without inositol, followed by incubation at 30°C for 1 day or 1.5 days (the <i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ mutant). The strains used were YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), carrying YEplac181 (vector), pKT1259 (p<i>CDC50</i>), pKT1788 (p<i>NEO1</i>), pKT1719 (p<i>ART5</i>), or pKT1754 (p<i>CHO1</i>).</p

Suppression of the defects in flippase mutants by depletion of inositol from culture medium.

<p>(A) Suppression of the growth defects. Cells were pregrown to early log phase in YPGA medium, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SD agar plate with or without inositol, followed by incubation at 30°C for 1.5 days. The strains used were YKT1066 (wild type, WT), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), YKT1909 (<i>P</i>_<i>GAL1</i><i>-NEO1 P</i>_<i>GAL1</i><i>-CDC50</i>), and YKT1887 (<i>P</i>_<i>GAL1</i><i>-NEO1 P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ). (B) Suppression of growth defects in the flippase null mutants. Cells were pregrown to early log phase in SD medium without inositol, and cell growth was examined as in (A), except that they were incubated at 30°C, 25°C, or 18°C for 1.5, 2, or 5 days, respectively. The strains used were YKT1066 (wild type, WT), YKT1944 (<i>cdc50</i>Δ), and YKT1945 (<i>cdc50</i>Δ <i>dnf1</i>Δ). YKT1945 was constructed by tetrad dissection of spores from a <i>cdc50</i>Δ/<i>CDC50 DNF1</i>/<i>dnf1</i>Δ heterozygous diploid on an inositol-depleted SD agar plate. (C) Suppression of the defects in endocytic recycling of Snc1p. Cells were grown in SD medium with or without inositol at 30°C for 12 h or 16 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with mRFP-Snc1p in polarized sites was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1912 (<i>mRFP-SNC1</i>, WT), YKT1936 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>mRFP-SNC1</i>), YKT1937 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>mRFP-SNC1</i>), and YKT1910 (<i>P</i>_<i>GAL1</i><i>-NEO1 mRFP-SNC1</i>). Bar: 5 μm. (D) Restoration of the plasma membrane location of GFP-Snc1p-pm. Cells were grown in SD-Ura or SD-Leu medium with or without inositol at 30°C for 16 h. More than 100 cells were microscopically observed, and the percent of cells with internally accumulated GFP-Snc1p-pm was determined. The strains used were YKT1066 (wild type, WT), YKT1120 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1660 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), carrying pKT1444 (pRS416-GFP-SNC1-pm) or pKT1491 (pRS315-GFP-SNC1-pm). Bar: 5 μm. (E) Formation of secretory vesicles. Cells were grown in SD with or without inositol at 30°C for 12 h, followed by a shift to 37°C for 2 h. Secretory vesicles were fractionated by the Nycodenz density gradient, and each fraction was measured for mRFP fluorescence intensity and the total amount of phospholipid phosphates. Strains used were YKT1844 (<i>sec6–4 mRFP1-Lact-C2</i>) and YKT1854 (<i>sec6–4 P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>mRFP1-Lact-C2</i>).</p

<i>S</i>. <i>cerevisiae</i> strains used in this study.

<p>YKT and KKT strains are isogenic derivatives of YEF473 and BY4743, respectively.</p><p>Only relevant genotypes are described.</p><p><i>S</i>. <i>cerevisiae</i> strains used in this study.</p

The growth defects of flippase mutants were suppressed by mutations in inositol transporters.

<p>(A) Suppression of the flippase mutations by overexpression was specific to <i>ART5</i>. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>, except that the <i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ) and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), both carrying YEplac195 (vector), pKT1720 (p<i>ART5</i>), pKT2135 (p<i>RIM8</i>), pKT2136 (p<i>ROD1</i>), pKT2137 (p<i>ROG3</i>), pKT1881 (p<i>ALY1</i>), pKT1882 (p<i>ALY2</i>), pKT2088 (p<i>ART5</i>-<i>PYm</i>), or pKT2138 (p<i>ART5</i>-<i>AMm</i>). YKT1511 and YKT1932 also carried pKT1263 (p<i>CDC50</i>) and pKT1469 (p<i>NEO1</i>), respectively, as positive controls. (B) Suppression of the growth defects of flippase mutants by mutations in inositol transporters. Cells were pregrown to early log phase in SGA-Ura or YPGA medium, and cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. The strains used were YKT1066 (wild type, WT), YKT1866 (<i>itr1</i>Δ <i>itr2</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1934 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ), YKT1935 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ <i>itr2</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1915 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ), YKT1914 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>itr1</i>Δ <i>itr2</i>Δ), YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), YKT1877 (<i>P</i>_<i>GAL1</i><i>-NEO1 itr1</i>Δ), and YKT1881 (<i>P</i>_<i>GAL1</i><i>-NEO1 itr1</i>Δ <i>itr2</i>Δ). YKT1529, YKT1513, and YKT1932 also carried YEplac195 (vector) or pKT1720 (p<i>ART5</i>).</p

Identification of <i>ART5</i> as a multicopy suppressor of flippase mutations.

<p>(A) Suppression of growth defects by overexpressing <i>ART5</i> in Cdc50p-depleted mutants carrying a mutation synthetically lethal with <i>cdc50</i>Δ. Cells were grown to early log phase in SGA-Ura medium, washed, and adjusted to a concentration of 2.5 × 10⁷ cells/ml. Drops of 4 μl from 5-fold serial dilutions were spotted onto a YPDA (Cdc50p-depleted) or YPGA (Cdc50p-expressed) agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1286 (<i>P</i>_<i>GAL1</i><i>-CDC50 gcs1</i>Δ), KKT116 (<i>P</i>_<i>GAL1</i><i>-CDC50 fpk1</i>Δ), and YKT1649 (<i>P</i>_<i>GAL1</i><i>-CDC50 neo1–101</i>), all carrying YEplac195 (vector), pKT1263 (p<i>CDC50</i>), or pKT1720 (p<i>ART5</i>). (B) Suppression of growth defects in flippase mutants by overexpression of <i>ART5</i>. Cells were grown and examined as in (A), except that the <i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ mutant was incubated at 25°C for 2 days. The strains used were YKT1511 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), all carrying each of the plasmids in (A), except YKT1932 carried pKT1469 (p<i>NEO1</i>) as a positive control. (C) Suppression of the defects in membrane trafficking in flippase mutants by overexpression of <i>ART5</i>. Cells were grown in YPDA medium at 25°C for 14 h (Cdc50p-depleted <i>crf1</i>Δ) or at 30°C for 12 h (Neo1p-depleted), followed by microscopic observation of small- or middle-budded cells. The percent of cells with polarized GFP- or mRFP-Snc1p was determined (n>100) and is shown with the mean ± standard deviation of three independent experiments. Representative images are shown. The strains used were YKT1933 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ <i>GFP-SNC1</i>) and YKT1910 (<i>P</i>_<i>GAL1</i><i>-NEO1 mRFP-SNC1</i>), both carrying each of the plasmids in (A), except YKT1910 carried pKT1469 (p<i>NEO1</i>) as a positive control. Bar: 5 μm. (D) Failure of <i>ART5</i> overexpression to suppress the alkylphosphocholine resistance and phospholipid-binding peptide sensitivity in a flippase mutant. Cells were grown to early log phase in SDA-Ura medium, washed, and adjusted to a concentration of 5.0 × 10⁶ cells/ml. Drops of 10 μl and 4 μl from 5-fold serial dilutions were spotted onto SDA-Ura containing 5.0 μg/ml miltefosine and YPDA containing 0.5 μg/ml papuamide B (pap B) or 2.0 μM duramycin agar plates, respectively, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (WT) and YKT715 (<i>lem3</i>Δ), both carrying YEplac195 (vector) or pKT1720 (p<i>ART5</i>).</p

Flippase mutations are not suppressed by activation of either the Ino2p-Ino4p pathway or the unfolded protein response.

<p>(A) Activation of the Ino2p-Ino4p pathway by the <i>opi1</i>Δ mutation does not suppress the growth defects of flippase mutants. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>, except the cells were incubated at 30°C or 25°C for 1 day or 2 days, respectively. The strains used were YKT38 (wild type, WT), YKT1938 (<i>opi1</i>Δ), YKT1510 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ), YKT1939 (<i>P</i>_<i>GAL1</i><i>-CDC50 crf1</i>Δ <i>opi1</i>Δ), YKT1120 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), YKT1940 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ <i>opi1</i>Δ), YKT1660 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), and YKT1941 (<i>P</i>_<i>GAL1</i><i>-NEO1 opi1</i>Δ). (B, C) Activation of the unfolded protein response pathway (UPR) does not suppress the growth defects of flippase mutants. (B) Expression of the UPR-activating <i>HAC1</i> (238 type S238A) mutant. Cells were grown in SG-Leu medium, washed, and adjusted to a concentration of 1.0 × 10⁷ cells/ml. Drops of 10 μl from 5-fold serial dilutions were spotted onto an SG-Leu, SD-Leu, or SD-Leu without inositol agar plate, followed by incubation at 30°C for 1.5 days. The strains used were KKT466 (<i>hac1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>), all carrying either YCplac111 (Vector) or pKT2139 [p<i>HAC1</i> (238 type S238A)]. (C) Treatment with TM or DTT. Cell growth was examined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120108#pone.0120108.g001" target="_blank">Fig. 1A</a>. Cells were spotted onto a YPGA, YPDA containing 0.5 μg/ml TM, or SD containing 6.5 mM DTT agar plate, followed by incubation at 30°C for 1 day. The strains used were YKT1066 (wild type, WT), KKT466 (<i>hac1</i>Δ), YKT1529 (<i>P</i>_<i>GAL1</i><i>-CDC50 dnf1</i>Δ <i>crf1</i>Δ), YKT1513 (<i>P</i>_<i>GAL1</i><i>-CDC50 lem3</i>Δ <i>crf1</i>Δ), and YKT1932 (<i>P</i>_<i>GAL1</i><i>-NEO1</i>).</p

Treatment with ligands for PPARβ/δ or PPARγ increase <i>CERS3</i> and <i>ELOVL4</i> mRNA expression in keratinocytes, in both a dose- and time-dependent manner.

<p>(A) Keratinocytes were incubated with either DMSO or activators of the indicated receptor, PPARβ/δ (10 µM L-165,041) or PPARγ (7.5 µM troglitazone), for the indicated times. (B) Keratinocytes were incubated for 24 h with the indicated concentrations of the PPARβ/δ activator L-165,041 or the PPARγ activator troglitazone. (A and B) Total RNA prepared from each culture was subjected to real-time PCR using primers specific for <i>ELOVL4</i> or <i>CERS3</i>, and for <i>GUSB</i> for standardization. The expression level of each mRNA was calculated by normalizing to that of <i>GUSB</i>. Values presented are the amount of the respective mRNA relative to that from cells harvested at 0 h (A) or from cells not treated with activator (B), and represent the mean ± S.D. from three independent experiments. Statistically significant differences to the samples at 0 h (A) or untreated samples (B) are indicated (*p<0.05, **p<0.01; Student’s t-test).</p

ELOVL4 is up-regulated during keratinocyte differentiation.

<p>(A) Total RNA was prepared from keratinocytes differentiated for 0, 3, or 6 days in differentiation medium. SYBR green-based real-time quantitative PCR was performed using primers specific for <i>ELOVL1</i>, <i>ELOVL4</i>, <i>ELOVL5, ELOVL6</i>, or <i>ELOVL7</i>, and for <i>GUSB</i> as an internal control. The expression level of each <i>ELOVL</i> mRNA was calculated using a standard curve and normalized to that of <i>GUSB</i>. Values presented are the amount of each <i>ELVOL</i> mRNA relative to that of <i>ELOVL4</i> at day 0, and represent the mean ± S.D. from three independent reactions. Statistically significant differences from day 0 are indicated (*p<0.05, **p<0.01, ***p<0.001; Student’s t-test). E1, <i>ELOVL1</i>; E4, <i>ELOVL4</i>; E5, <i>ELOVL5</i>; E6, <i>ELOVL6</i>; E7, <i>ELOVL7</i>. (B) Total cell lysates (10 µg protein) prepared from keratinocytes differentiated for 0, 2, 4, or 8 days in differentiation medium were subjected to immunoblotting with an anti-CERS3 antibody, anti-CERS2 antibody, anti-involucrin antibody, or, to demonstrate uniform protein loading, anti-GAPDH antibody. (C) Total membrane proteins (40 µg protein) from keratinocytes differentiated for 0, 2, or 4 days were incubated with the indicated acyl-CoA (50 µM) and 0.075 µCi [¹⁴C] malonyl-CoA for 30 min at 37°C. After termination of the reactions, acyl-CoAs were converted to FAs and separated by normal-phase TLC, followed by detection and quantification by a BAS-2500 bioimaging analyzer (Fuji Photo Film). Values presented are FA levels and represent the mean ± S.D. from three independent experiments. Statistically significant differences compared to 0 day cells are indicated (*p<0.05; Student’s t-test).</p

Activation of PPARβ/δ, PPARγ, or LXR increases <i>CERS3, ELOVL4,</i> and <i>ELOVL7</i> mRNA expression in keratinocytes.

<p>Keratinocytes were incubated for 24 h with vehicle (DMSO) or with an activator of the indicated transcription factor: LXR (10 µM TO901317), RAR (1 µM all-<i>trans</i>-retinoic acid), RXR (1 µM 9-<i>cis</i>-retinoic acid), PPARα (10 µM WY14643), PPARβ/δ (10 µM L-165,041), PPARγ (7.5 µM troglitazone), or vitamin D receptor (VDR) (0.1 µM 1α, 25-dihydroxyvitamin D3). Total RNA prepared from each culture was subjected to real-time quantitative PCR using primers specific for <i>ELOVL1</i>, <i>ELOVL4</i>, <i>ELOVL6</i>, <i>ELOVL7</i>, <i>CERS2</i>, or <i>CERS3</i> and for <i>GUSB</i> for standardization. The expression level of each mRNA was calculated by normalizing to that of <i>GUSB</i>. Values presented are the amount of the respective mRNA relative to that from cells treated with vehicle, and represent the mean ± S.D. from three independent experiments. Statistically significant differences to DMSO controls are indicated (*p<0.05; **p<0.01; ***p<0.001; Student’s t-test).</p