MIER1α is localized in the nucleus in ER- breast carcinoma cells.

<p>MDA231-derived cell lines, VC5 (vector) and MC2 (stably expressing ERα), were transfected with myc-tagged MIER1α and analyzed by confocal microscopy using DAPI (a, e), 9E10 anti-myc tag (b,f), anti-ERα (c,g) and the secondary antibodies described in the legend to Fig. 1. Panel d shows merged MIER1α and DAPI staining while panel h shows merged MIER1α and ERα staining. Note that MIER1α is localized in the nucleus in VC5 cells, even in the absence of ERα (arrowheads in panels a-d). (B) Histogram showing the results of 3 independent experiments; random fields were selected and the staining pattern of each cell within the field was scored visually. 170-380 cells were scored for each cell line. Plotted is the percentage of cells in each category ± S.D; there is no significant difference between the percent nuclear for the two cell lines (p>0.05).</p

HDAC1 and 2 knockdown reduces nuclear localization of MIER1α.

<p>MCF7 cells were transfected with myc-tagged MIER1α and either control shRNA or HDAC shRNA(s), then analyzed by immunoblotting (A–B) or confocal microscopy (C–E). (A–B) Western blot analysis to confirm HDAC knockdown and specificity of each shRNA. Extracts from MCF7 cells were transfected with myc-tagged MIER1α and either control scrambled shRNA (Con; lane 1, 5), HDAC1 shRNA (Hd1; lane 2, 6), HDAC2 shRNA (Hd2; lane 3, 7) or both HDAC1 and 2 shRNAs (Hd1+Hd2; lanes 4, 8). Duplicate samples were stained with anti-HDAC1 (lanes 1–4) or anti-HDAC2 (lanes 5–8). The blots were restained with anti-β-actin (lower panels) to verify equal loading. (B) The HDAC protein bands shown in (A) were quantified using ImageJ, normalized to β-actin and plotted as a proportion of the HDAC level in control cells. Note that each shRNA is specific for its target. (C) Localization was analyzed in parallel samples by confocal microscopy. Histogram showing the results of 2 independent experiments; random fields were selected and the staining pattern of each cell within the field was scored visually; 400–600 cells were scored for each shRNA. Plotted are the percentage of cells in each category ± S.D; the percent nuclear of HDAC1, HDAC2 or HDAC1&2 depleted cells were significantly less than that of controls (p<0.05). (D) Bar graph showing the intracellular distribution of MIER1α. Pixel values for the nuclear and the cytoplasmic compartments were measured in confocal z-stacks using Image J v1.38; plotted is the proportion of the total signal in each compartment, using measurements from 20–25 cells for each shRNA. The proportion of MIER1α in the nucleus is significantly less in depleted cells than in controls (p<0.05 for each). (E) Illustrative examples of cells depleted for both HDAC1 and 2, stained as described in the legend to Fig. 5 for MIER1α (panels b, f) and with combined anti-HDAC1 and 2 antibodies (panels c, g), using MCF7 cells co-transfected with myc-tagged MIER1α and either control shRNA (panels a-d) or HDAC1 + HDAC2 shRNAs (panels e-h). Note that MIER1α staining is nuclear in control cells (arrowheads in a–d) but predominantly ‘whole cell’ in cells with reduced HDAC1&2 staining (arrowheads in e–h).</p

Nuclear localization requires an intact ELM2 domain.

<p>MCF7 cells were transfected with a myc-tagged intact ELM2 domain (aa164–283) (panels a-c) or a myc-tagged ELM2 deletion construct containing aa164-239 (panels d-f), aa240-283 (panels g-i), aa164-251 (panels j-l), aa164-273 (panels m-o). Localization was analyzed by confocal microscopy using DAPI and the 9E10 anti-myc tag antibody. (A) Illustrative examples of stained cells showing MIER1α localization. Note that nuclear localization was only detected with an intact ELM2 domain (a-c); arrowheads indicate examples of stained nuclei. The rest of constructs displayed whole cell staining (arrows in d-o). A schematic, drawn to scale and illustrating the constructs used, is shown on the right as are the amino acids (aa) encoded by each construct. The myc epitope tag is shown in green. (B) Histogram showing the results of 3 independent experiments; random fields were selected and the staining pattern of each cell within the field was scored visually. 465-565 cells were scored for each construct. Plotted is the percentage of cells in each category ± S.D; the percent nuclear of all deletion constructs are significantly less than that of the intact ELM2 domain (p<0.05).</p

Additional file 6: Table S5. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

Differentially expressed proteins with no annotated functions at H1d when compared to E19d in chicken embryos. (DOCX 32 kb

Additional file 2: Table S1. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

Differentially expressed proteins at E19d when compared to E14d in chicken embryos. (DOCX 84 kb

Rationally Designed Calcium Phosphate/Small Gold Nanorod Assemblies Using Poly(acrylic acid calcium salt) Nanospheres as Templates for Chemo-photothermal Combined Cancer Therapy

Elaborately designed novel multifunctional therapeutic agents are highly desired for efficient cancer therapy. In this work, a new therapeutic nanoplatform based on calcium phosphate/small gold nanorod assemblies modified with methoxy-poly­(ethylene glycol)-thiol (designated as PEGylated CaP/Au NR assemblies) is created via a mild, reproducible, and simple route for the first time. The obtained PEGylated CaP/Au NR assemblies possess many virtues including outstanding drug-loading capacity, excellent photothermal conversion efficiency (η, ∼38.5%), pH/near-infrared (NIR) dual-responsive release property, and good biocompatibility. After loading doxorubicin (DOX) in PEGylated CaP/Au NR assemblies, the DOX-loaded PEGylated CaP/Au NR assemblies can simultaneously supply intense heating effect and increased DOX release under 808 nm NIR laser, achieving excellent antitumor therapeutic effect in vitro and in vivo. Furthermore, the combination of DOX-loading and photothermal treatment upon PEGylated CaP/Au NR assemblies displays better therapeutic effect than single chemotherapy or photothermal therapy. Furthermore, the comprehensive methyl thiazolyl tetrazolium (MTT), hemolysis, and histological assays manifest no obvious toxicity of PEGylated CaP/Au NR assemblies. Our work elucidates the great prospect of PEGylated CaP/Au NR assemblies as a therapeutic agent for synergistic chemo-photothermal cancer therapy

Additional file 10: Table S9. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

Differentially expressed proteins which were not assigned to any known functions at H1d when compared to E19d in chicken embryos. (DOCX 28 kb

Additional file 3: Table S2. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

Differentially expressed proteins at H1d when compared to E19d in chicken embryos. (DOCX 68 kb

Additional file 1: Figure S1. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

The distribution of peptide length, peptide number, protein mass and protein’s sequences coverage. (DOCX 243 kb

Additional file 7: Table S6. of Proteomics reveals changes in hepatic proteins during chicken embryonic development: an alternative model to study human obesity

Detailed pathway enrichment of differential expression protein between E14d and E19d in chicken embryos. (DOCX 22 kb