DS_10.1369_0022155418788845 – Supplemental material for Expression of Luminal Progenitor Marker CD117 in the Human Breast Gland

<p>Supplemental material, DS_10.1369_0022155418788845 for Expression of Luminal Progenitor Marker CD117 in the Human Breast Gland by Jiyoung Kim and René Villadsen in Journal of Histochemistry & Cytochemistry</p

Number and mean plaque size in mock- or Tat-virus-injected APP/PS1 mouse hippocampi.

<p>Lentiviruses containing an expression construct for mock or Tat were injected stereotaxically into the hippocampus of APP/PS1 mice. After 4 months, sections of brain were stained with Aβ antibody and the number and size of plaques were determined using Axiovision software (Carl Zeiss). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077972#s2" target="_blank">Results</a> represent the mean ± s.d.</p

HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

<div><p>HIV-1 Tat protein plays various roles in virus proliferation and in the regulation of numerous host cell functions. Accumulating evidence suggests that HIV-1 Tat also plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting intracellular communication. Amyloid beta (Aβ) is generated from amyloid precursor protein (APP) and accumulates in the senile plaques of Alzheimer's disease patients. This study demonstrates that Tat interacts with APP both <i>in vitro</i> and <i>in vivo</i>, and increases the level of Aβ42 by recruiting APP into lipid rafts. Co-localization of Tat with APP in the cytosol was observed in U-87 MG cells that expressed high levels of Tat, and redistribution of APP into lipid rafts, a site of increased β- and γ-secretase activity, was demonstrated by discontinuous sucrose density gradient ultracentrifugation in the presence of Tat. Furthermore, Tat enhanced the cleavage of APP by β-secretase <i>in vitro</i>, resulting in 5.5-fold higher levels of Aβ42. This was consistent with increased levels of β-C-terminal fragment (β-CTF) and reduced levels of α-CTF. Moreover, stereotaxic injection of a lentiviral Tat expression construct into the hippocampus of APP/presenilin-1 (PS1) transgenic mice resulted in increased Tat-mediated production and processing of Aβ <i>in vivo</i>. Increased levels of Aβ42, as well as an increase in the number and size of Aβ plaques, were observed in the hippocampus following injection of Tat virus compared with mock virus. These results suggest that HIV-1 Tat may contribute to HAND by interacting with and modifying APP processing, thereby increasing Aβ production.</p></div

HIV-1 Tat promotes localization of APP to lipid rafts.

<p>(<i>A</i>) The amounts of Triton X-100-resistant APP increased in Lenti-Tat infected U-87 MG cells. U-87 MG cells were transduced with mock virus or with Lenti-Tat virus, and incubated for 20 days. Cells were harvested and lysed in the presence of 1% Triton X-100 and the soluble and insoluble fractions were separated by centrifugation. WCE; whole cell extracts, Sol; soluble fraction, Insol; insoluble fraction. (<i>B</i>) U-87 MG cells were transduced with mock virus or Lenti-Tat virus and incubated for 14 days. Cells were harvested and lysed in the presence of 1% Triton X-100 and subjected to 5% and 35% discontinuous sucrose density gradient ultracentrifugation. Fractions of 0.5 ml were harvested from the top to the bottom and analyzed by Western blotting for APP, flotillin-1, transferrin receptor and Tat. The majority of APP was found in the non-raft fractions in mock –infected cells (Left panel) while large amount of APP was moved to lipid raft fractions in Lenti-Tat infected cells (Right panel).</p

HIV-1 Tat increases Aβ levels and the number and size of amyloid plaques in the APP/PS1 mouse hippocampus.

<p>Mock (<i>A</i>) or syn-Tat (<i>B</i>) was injected stereotaxically into APP/PS1 mouse hippocampi. At 2 months after injection, half of the brain was frozen-sectioned and stained with anti-Tat antibody, and then visualized under an inverted phase fluorescence microscope. Scale bar = 50 µm. (<i>C</i>) Mouse hippocampi (n = 9) injected with mock (syn) or Tat (syn-Tat) were dissected, homogenized 4 months after injection, and Aβ42 levels in the hippocampus extracts were examined by ELISA. The Aβ42 concentration for each mouse is shown on the y-axis, and the injected virus is indicated on the x-axis. (<i>D</i>) Equal volumes of syn-Tat and Lenti-Tat were mixed and injected unilaterally into the mouse hippocampus (n = 5). After 4 months, the hippocampus was dissected from mock (con)- and mixed Tat (Tat)-virus-injected hemispheres, and Aβ42 levels were investigated by ELISA. Aβ42 levels were 1.6-fold higher in the Tat-expressing hemisphere of mouse hippocampi (P<0.001). (<i>E</i>) Representative Western blots for the detection of Aβ. Twenty micrograms of mouse hippocampus extract were separated on 16.5% Tris-Tricine PAGE gels followed by Western blotting with anti-APP (6E10) antibody. Five micrograms of hippocampus extract were used for Western blotting for APP and actin. (<i>F</i>, <i>G</i>) Representative photographs of the Aβ plaque burden in APP/PS1 mice are shown. Brain sections of APP/PS1 mice injected with mock (<i>F</i>) or Lenti-Tat (<i>G</i>) virus were stained with anti-Aβ antibody (6E10). Scale bar = 50 µm.</p

HIV-1 Tat interacts with APP both <i>in vitro</i> and <i>in vivo</i>.

<p>(<i>A</i>) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). (<i>B</i>) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. (<i>C</i>) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. (<i>D</i>) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. (<i>E</i>) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. (<i>F</i>) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.</p

HIV-1 Tat alters the processing of APP and increases the levels of β-CTF and Aβ42.

<p>(<i>A</i>) Level of β-CFT was increased in Lenti-Tat, or Lenti-mTat infected cells while the amount of total APP was not changed. U-87 MG cells were transduced with mock, Lenti-Tat, or Lenti-mTat virus and incubated for 14 days. The cell number was counted, and equal numbers of cells from each sample were analyzed in 16.5% Tris-Tricine gels to detect CTF or 8% SDS-PAGE for the detection of APP and β-actin. Both α- and β-CTF were detected with an anti-APP C-terminal antibody (A8717, top panel), and β-CTF was detected with 6E10 (upper middle panel). The total amounts of APP (22C11, lower middle panel) and β-actin (bottom panel) were not changed by the expression of Tat or mutant Tat protein. (<i>B</i>) Aβ42 level was increased in Lenti-Tat and Lenti-mTat infected U-87 MG cells. Average of Aβ42 concentration for 9 days was calculated from individual Aβ42 concentration. The Aβ42 concentrations in Lenti-Tat- or Lenti-mTat-infected cells were increased by 5.58±0.83 (mean ± SE, P<0.005)-fold or 3.64±0.38 (mean ± SE, P<0.005)-fold, respectively, compared with mock-infected cells. (<i>C</i>) Treatment with the neprilysin inhibitor thiorphan further increased the level of Aβ42 in the presence of Tat. Mock or Lenti-Tat-infected U-87 MG cells were cultured for 3 days in the absence or presence of 10 µM thiorphan. Culture supernatant was harvested from each cell culture and analyzed by Aβ42 ELISA. Error bars represent the mean ± SD. *P<0.05, **P<0.001.</p

HIV-1 Tat colocalizes with APP in U-87 MG cells.

<p>Fluorescence microscopy images of Tat-transfected U-87 MG cells immunostained with anti-Tat and anti-APP (A8717) antibodies are shown. U-87 MG cells were transfected with the wild-type Tat construct and incubated for 16 h. The cells were fixed and stained with anti-Tat or anti-APP antibodies followed by FITC-conjugated anti-mouse or rhodamine-conjugated anti-rabbit antibodies, respectively. Nuclear (<i>A</i>) and nuclear plus cytosolic (<i>B</i> and <i>C</i>) localization of Tat is shown. Scale bar = 10 µm.</p

The most significantly different metabolites identified in the strain lacking CIR1 relative to the wild-type.

a<p>Rt, retention time.</p>b<p>Level of significance (<i>p</i>-value <0.005) of the difference between strains tested.</p>c<p>Number of hydrogen atoms derivatized.</p

The <i>cir1</i> mutant displayed decreased sensitivity to azole antifungal drugs.

<p>The growth of the wild-type (WT) or the <i>cir1</i> mutant (<i>cir1Δ</i>) in media containing an antifungal drug was monitored. Ten-fold serial dilutions of cells (starting at 10⁴ cells) were spotted onto YPD plates with the drug indicated. Plates were incubated at 30°C for 2 days.</p