Identification of <i>S. pombe</i> genes differentially expressed with respect to genotype.

<p>Volcano plot (left panels) and scatter plot (right panels) analysis of the expression of all <i>S. pombe</i> genes in DMSO (<b>A</b>) or LatA (<b>B</b>) treated cells of the indicated genotype. Horizontal green lines in volcano plots represent a p-value of 0.05. Vertical green lines in volcano plots represent threshold for a 1.5 fold change in expression. Red squares in volcano plots indicate differentially expressed genes. Diagonal green lines in scatter plots represent the threshold for a 1.5 fold change in expression. The color of the squares in scatter plots indicates the level of expression of that gene in DMSO (<b>A</b>) or LatA (<b>B</b>) treated wild-type cells.</p

Hif2p, Set3p, and Snt1p form a nuclear-localized complex.

<p>(<b>A</b>) Cells expressing the indicated fusion proteins were grown to mid-log phase at 30°C in YES media, fixed, and then stained with DAPI and observed using the DAPI and GFP filter sets. Bar, 5 µm. (<b>B</b>) Cells expressing the indicated fusion proteins were grown to mid-log phase in YES, lysed under native conditions, and subjected to anti-Myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the HA epitope.</p

Domain structure of Set3p, Snt1p, and Hif2p.

<p>Structures are based upon Uniprot database predictions. Arabic numerals to the right of each schematic indicate the length of the protein in amino acids. Arabic numerals below each schematic indicate the amino acid position of that domain within the protein. Schematics are not drawn to scale.</p

<i>hif2</i>, <i>set3</i>, and <i>snt1</i> deletion mutants are hyper-sensitive to LatA treatment.

<p>(<b>A</b>) Ten-fold serial dilutions of logarithmically growing cells of the indicated genotype were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) at 30°C for 3 d. (<b>B</b>) Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.5 µM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µm. (<b>C</b>) Quantitation of phenotypes of cells treated as in B. Between 200 and 500 cells were counted for each genotypic class.</p

<i>set3Δ</i> mutants are impaired in their ability mount a proper transcriptional response to LatA treatment.

<p>(<b>A</b>) Volcano plot analysis of the expression of genes annotated by GO as having a cellular response to stress. The response of wild-type cells (left panel) and <i>set3Δ</i> mutants (right panel) treated with LatA are shown. Horizontal green lines represent a p-value of 0.05. Vertical green lines represent the threshold for a 1.5 fold change in expression. Red squares indicate differentially expressed genes. (<b>B</b>) Scatter plot analysis comparing the expression of stress response genes (both CESR genes and genes annotated by GO as having a cellular response to stress) in response to LatA treatment. Blue squares indicates data points for wild type cells. Red diamonds indicate data points for <i>set3Δ</i> mutants. Genes up-regulated in response to LatA in wild-type cells are highlighted with a blue oval. Genes down-regulated in response to LatA in wild-type cells are highlighted with a pink oval.</p

The core environmental stress response genes (CESR) respond to LatA treatment in wild-type cells.

<p>Volcano plot analysis of the expression of the CESR genes in wild-type strains treated with 0.5 µM LatA. Blue diamonds represent CESR genes normally up-regulated in response to multiple stresses. Red squares represent CESR genes normally down-regulated in response to multiple stresses.</p

Set3p, Snt1p and Hif2p levels increase two- to three-fold upon treatment with low doses of LatA.

<p>(<b>A</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA (left panel) or 1 µM LatA (right panel). Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin was used as a loading control. (<b>B and C</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA. Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin or the RNA pol II carboxy-terminal domain (CTD) were used as loading controls. Signal intensities relative to the loading controls of three independent trials were quantified using ImageJ 1.41 Gel Analyzer software and are plotted below representative blots. Error bars indicate sd.</p

The expression levels of cytokinesis related genes are not differentially regulated.

<p>(<b>A</b>) Cells of the indicated genotypes were grown to mid-log phase at 30°C in YES, and then treated with DMSO (<b>A</b>) or 0.5 µM LatA (<b>B</b>) for 3 hours. Total RNA was extracted and used in expression profiling using Affymetrix Yeast 2.0. Genechips. Graphs shows scatter plot analysis comparing the expression of cytokinesis related genes in wild-type and <i>set3Δ</i> strains. Green lines represent the threshold for a 1.5 fold change in transcript levels. The color of the squares indicates the level of expression of that gene in DMSO (<b>A</b>) or LatA (<b>B</b>) treated wild-type cells.</p

<i>set3</i> functions in a pathway parallel to those defined by <i>clp1</i> and <i>lsk1</i>.

<p>Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.1 µM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Two hundred cells were counted for each genotypic class.</p

Coating Nanoparticles with Plant-Produced Transferrin–Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

The encapsulation of drugs to nanoparticles may offer a solution for targeted delivery. Here, we set out to engineer a self-assembling targeting ligand by combining the functional properties of human transferrin and fungal hydrophobins in a single fusion protein. We showed that human transferrin can be expressed in <i>Nicotiana benthamiana</i> plants as a fusion with <i>Trichoderma reesei</i> hydrophobins HFBI, HFBII, or HFBIV. Transferrin–HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of-concept for the functionalization of hydrophobin coatings with transferrin as a targeting ligand