Additional file 2: of Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method

Sequential 2D images of X-Y cross-sections of the EGFP-positive antral follicle. Female EGFP-transgenic mice were transcardially perfused with 4% PFA containing PI. After the isolated ovaries were subjected to CUBIC, sequential 2D images of X-Y cross-sections containing an antral follicle corresponding to Fig. 3e were taken using a confocal microscope. (MOV 710 kb

Additional file 1: of Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method

Sequential 2D images of X-Y cross-sections of the EGFP-positive preantral follicles. Female EGFP-transgenic mice were transcardially perfused with 4% PFA containing PI. After the isolated ovaries were subjected to CUBIC, sequential 2D images of X-Y cross-sections containing primordial and preantral follicles corresponding to Fig. 3d were taken using a confocal microscope. (MOV 918 kb

Additional file 3: of Three-dimensional evaluation of murine ovarian follicles using a modified CUBIC tissue clearing method

GFP immunohistochemistry using ovaries of CAG-EGFP mice. Female CAG-EGFP mice were fixed with the transcardial perfusion of 4% PFA. Sections were stained with Hoechst 33,342 and anti-GFP antibody to reveal the expression of EGFP protein. Note that although GFP fluorescence was undetectable in granulosa cells, the expression of immunoreactive EGFP protein was detected. Scale bars, 100 Οm. ( 668 kb

Reactivity of lectins against proteins in the mouse testicular TS fraction co-immunoprecipitated with Ts4.

<p>The immunoprecipitated proteins from testicular TS fraction with either Ts4 or normal control IgM (n.c.) were separated by SDS-PAGE under reducing conditions. Control experiments were conducted under the same conditions except for the absence of the TS fraction (buf). The testicular TS fraction was used as a positive control (IP (-)). Proteins were electroblotted onto PVDF membranes and then probed with E-PHA (A), PSA (B), WGA (C), DSA (D), L-PHA (E), DBA (F), or SJA (G). Arrowheads indicate the lectin-reactive bands corresponding to TEX101. Mr, molecular mass.</p

The MS² spectra of main glycans [dHex1Hex3HexNAc5 (1) in S1A and S1B Fig].

<p>The 38-kDa band (A) and 70-kDa band (B). The potential deduced structures of <i>N</i>-glycan as the Ts4-epitope (C). Bisecting GlcNAc (i), LacdiNAc (ii). Green circle: mannose, red triangle: fucose, blue square: GlcNAc, yellow square: GalNAc.</p

Immunoreactivity of Ts4 against testicular proteins pretreated with periodic acid.

<p>The testicular TS fractions (each 5 μg protein) were loaded on a 10% gel, separated by SDS-PAGE under reducing conditions, and then blotted onto a PVDF membrane. The PVDF membrane was divided into individual lanes, which were treated with 0.075 M NaIO₄ and HIO₄ 2H₂O in PBS for various times. Specific bands were detected with Ts4 or 6035 (arrowheads). Mr, molecular mass.</p

A Functionally Superior Second-Generation Vector Expressing an Aurora Kinase-A-Specific T-Cell Receptor for Anti-Leukaemia Adoptive Immunotherapy

<div><p>Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient’s own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this ‘siTCR’ vector. We then compared the activity of this vector against the original, ‘conventional’ vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.</p></div

Profiles of expression patterns of Ts4-reactive molecule(s) and TEX101 in male and female germ cells, embryonic cells, and somatic cells by immunomorphological analysis ^a.

<p>^a Data from references (2, 3).</p><p>^b Interstitial cells include leydig cells.</p><p>+ Positive,</p><p>–Negative.</p><p>* Cells that possess Ts4-reactive molecules but not TEX101.</p><p>Profiles of expression patterns of Ts4-reactive molecule(s) and TEX101 in male and female germ cells, embryonic cells, and somatic cells by immunomorphological analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133784#t001fn001" target="_blank">^a</a>.</p

The total ion current chromatogram of <i>N</i>-glycans from the 38-kDa band obtained by FT-ICR-MS (<i>m/z</i> 700–2,000).

<p>The positive-ion mode (A) and the negative-ion mode (B). Green circle: mannose, white circle: galactose, red triangle: fucose, blue square: GlcNAc, yellow square: GalNAc, pink diamond: <i>N</i>-acetylneuraminic acid (NeuNAc), black asterisk: glycan carrying bisecting GlcNAc or LacdiNAc, gray asterisk: non-glycan peak.</p

Binding of AURKA tetramer by transduced CD8+ effector cell populations.

<p>CD8+ cells from three donors were transduced with siAUK or coAUK vectors, at a range of MOIs (vector supernatant applied to Retronectin-coated plates at 25%, 50%, and 100% of usual volume, diluted where appropriate with fresh culture medium). Binding of fluorophore-conjugated AUK_207-215/HLA-A*0201 tetramer was analysed by flow cytometry. Genomic DNA was also collected, and vector copy number determined by qPCR. (A) Tetramer-binding and vector copy number were compared for the population as a whole. Correlation coefficients were compared using Preacher’s calculation (p<0.05). (B) Cells were also labelled for CD8+, and Vβ12 transgene expression. Cells from each MOI sample were gated for CD8 and Vβ12 positivity, and the percentage of tetramer-binding cells was recorded.</p