Detailed characterization of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A: CHO cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by luciferase reporter expression. The luminescence of reporter-only transfected cells (1.8±0.3%; n = 15) was subtracted from all values. B: CHO cells were transfected with 100 ng each of NE+EC and cultured in the presence of different concentrations of 4OHT. Luminescence in A and B was normalized to the luminescence observed in N+C transfected cells (100 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. C: PC12 20.4 cells were transfected with different amounts of NE+EC and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Recombination was analyzed by EGFP reporter expression using flow cytometry. The percentage of EGFP⁺-cells observed in the absence of NE+EC (4.4±0.7%; n = 9) was subtracted from all values. D: Cells were transfected with NE+EC plasmids (400 ng each/well) and cultured in the presence of different concentrations of 4OHT. The number of EGFP-positive cells in C and D was normalized to the number of EGFP-positive cells in N+C transfected cultures (400 ng of each plasmid/well) in the absence of 4OHT, which was set as 100%. All panels summarize data from a minimum of 3 independent experiments, each of which was performed at least in triplicate.</p

Immunocytochemical analysis of the split-CreERT2 protein combination NCre-ERT2 + ERT2-CCre (NE+EC).

<p>A, B: CHO cells cultured in 24-well cell culture plates were transfected with NE+EC (150 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and an expression vector coding for nuclear DsRed (100 ng/well; shown in red). Cells were cultured in the absence (A) or presence of 4OHT (1 µM, B). Recombination is detected by EGFP reporter expression (green; visualized by immunostaining using anti-GFP-antibodies). In blue, DAPI-staining of all nuclei is shown. C–F: Immunocytochemical confirmation of expression of NE and EC and of recombination in NE+EC transfected cells after application of 4OHT. CHO cells were transfected with NE+EC (400 ng of each plasmid/well) along with a reporter plasmid which expresses EGFP only after Cre-mediated DNA recombination (400 ng/well) and cultured in the presence of 4OHT (1 µM). NE (C), EC (D) and EGFP (E) were visualized by immunostaining for Flag-tag, Myc-tag or GFP, respectively, and detected by Cy5-, Cy3- or Cy2-conjugated secondary antibodies. F shows the merged images with NE in magenta, EC in red, EGFP in green and DAPI in blue. Note the additional nuclei stained with DAPI but not by anti-Flag- or anti-Myc-antibodies, which are also negative for EGFP. The bar in F corresponds to 20 µm and applies to all panels.</p

Western Blot analysis of the expression of the different split-CreERT2 constructs.

<p>CHO cells were transfected with the different combinations of split-CreERT2 plasmids as indicated. Expression of N, NE and EN was analyzed by immunoblotting with anti-Flag antibodies, whereas C, CE and EC were detected by anti-Myc as well as anti-Cre antibodies. GAPDH was used as a loading control. The observed sizes of the proteins were as expected (N: 14.8 kDa; NE: 50.1 kDa; EN: 51.1 kDa; C: 41.6 kDa; CE: 77.0 kDa; EC: 78.0 kDa; GAPDH: 35.9 kDa).</p

Split-CreERT2 mediated DNA recombination analyzed in CHO cells by luciferase assays.

<p>A: CHO cells cultured in 96-well cell culture plates were transfected with the different combinations of split-CreERT2 plasmids (for abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone-0008354-g001" target="_blank">Fig. 1</a>; 100 ng of each plasmid/well) and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). Cre-dependent DNA recombination was analyzed by cotransfection of a CMV-LoxP-STOP-LoxP-luciferase reporter plasmid. Luciferase activity was normalized to luciferase activity in N+C transfected cells cultured in the absence of 4OHT. B: Induction ratios were calculated as [(luciferase activity in the presence of 4OHT)/(luciferase activity in the absence of 4OHT)]. The dotted line denotes a ratio of 1, which means no induction. C: A functional index of each combination of split-CreERT2 proteins was calculated as [(induction ratio)×(luciferase activity in the presence of 4OHT)]. This functional index is highest if the combination of split-CreERT2 proteins shows high inducibility and high activity in the presence of 4OHT. The figure shows data from 3 independent experiments, each of which was performed at least in triplicate. n.c.: not calculated.</p

Split-CreERT2 mediated DNA recombination analyzed in PC12 20.4 cells by flow cytometry.

<p>A: PC12 20.4 cells, which harbor a stably transfected CMV-LoxP-STOP-LoxP-EGFP reporter cassette, were cultured in 24-well cell culture plates and transfected with the different combinations of split-CreERT2 plasmids (for abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone-0008354-g001" target="_blank">Fig. 1</a>; 400 ng of each plasmid/well) and cultured in the absence (control, open bars) or presence of 4OHT (1 µM, black bars). B: Induction ratios were calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone-0008354-g002" target="_blank">Fig. 2</a>. The dotted line denotes a ratio of 1, which means no induction. C: A functional index of each combination of split-CreERT2 proteins was calculated as indicated in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone-0008354-g002" target="_blank">Fig. 2</a>. The figure shows data from 8 independent experiments, each of which was performed at least in triplicate. n.c.: not calculated.</p

Split-CreERT2 constructs.

<p>The original, non-inducible split-Cre proteins NCre and CCre have recently been described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008354#pone.0008354-Hirrlinger1" target="_blank">[15]</a>. To generate NCre-ERT2 and CCre-ERT2, the ERT2-domain was fused to the C-terminus of NCre and CCre, respectively. Vice versa, the ERT2-domain was fused to the N-terminus of NCre and CCre to obtain ERT2-NCre and ERT2-CCre, respectively. The abbreviations for the different constructs used in this paper are given in brackets. The thick vertical line at the N-terminus of all constructs denotes the position of the corresponding immunotag (NCre containing constructs: Flag-tag; CCre containing constructs: Myc-tag).</p

Atomic Force Microscopy and Analytical Ultracentrifugation for Probing Nanomaterial Protein Interactions

Upon contact with the human body, nanomaterials are known to interact with the physiological surroundings, especially with proteins. In this context, we explored analytical methods to provide biologically relevant information, in particular for manufactured nanomaterials as produced by the chemical industry. For this purpose, we selected two batches of SiO₂ nanoparticles as well as four batches of CeO₂ nanoparticles, each of comparably high chemical purity and similar physicochemical properties. Adsorption of serum proteins and bovine serum albumin (BSA) was quantified by SDS-PAGE in combination with densitometry and further investigated by atomic force microscopy (AFM) and analytical ultracentrifugation (AUC). The protein adsorption to SiO₂ nanoparticles was below the limit of detection, regardless of adjusting pH or osmolality to physiological conditions. In contrast, the four CeO₂ nanomaterials could be classified in two groups according to half-maximal protein adsorption. Measuring the work of adhesion and indention by AFM for the BSA-binding CeO₂ nanomaterials revealed the same classification, pointing to alterations in shape of the adsorbed protein. The same trend was also reflected in the agglomeration behavior/dispersibility of the four CeO₂ nanomaterials as revealed by AUC. We conclude that even small differences in physicochemical particle properties may nevertheless lead to differences in protein adsorption, possibly implicating a different disposition and other biological responses in the human body. Advanced analytical methods such as AFM and AUC may provide valuable additional information in this context

Relative expression of Kir4.1, aquaporin-4 and GFAP mRNA in wild-type and β2^−/−/γ3^−/− retina.

<p>Values are normalized to GAPDH expression and were set as 1 for wild-types (n = 9 animals; white bars). In retinal tissue of β2^−/−/γ3^−/− (n = 7 animals; black bars) the expression of Kir4.1 mRNA is significantly reduced (* P<0.05), while aquaporin-4 mRNA only shows a non-significant tendency to lower levels (ns: not significant; P = 0.597). In contrast, the expression level of GFAP, a marker for retinal gliosis, is upregulated (**P<0.01).</p

Immunocytochemical staining of acutely isolated Müller cells from β2, γ3 laminin knockout mice.

<p>β2, γ3 laminin knockout cells (β2^−/−/γ3^−/−) are shown in panels D–F and J–L; wild-type controls in A–C and G–I. The expression of Kir4.1 (B, E) and aquaporin-4 (H, K) was analysed in glutamine synthetase (GS)-positive Müller cells (A, D, G, J). C, F, I, L show overlays of GS (green) and Kir4.1 (red, C, F) or aquaporin-4 (red, I, L) staining, respectively. Endfeet of the cells are marked by arrows. The staining was quantified as described in the methods. In Müller cells from β2^−/−/γ3^−/− retinae the overall level of Kir4.1 staining (M) and of aquaporin-4 staining (N) quantified in whole cells was significantly lower as compared to wild-type Müller cells wild-types (wild-type: white bars; β2^−/−/γ3^−/−: black bars). These differences are mainly due to highly significant changes in endfeet expression levels of both proteins (M, N). Numbers of cells analysed was between 22 and 28 for wild-types and between 30 and 66 for β2^−/−/γ3^−/− mice; *** P≤0.001, ** P<0.01.</p

Immunohistochemical analysis of retinae of β2, γ3 laminin knockout mice.

<p>Immunohistochemical stainings of fixed retinal slices (A–C, E–F) or wholemounts (D) from wild-type (wt, A–F) and β2, γ3 laminin knockout mice (β2^−/−-/γ3^−/−, A′–F′, D″) are shown. A, A′: Staining for the Müller cell filament protein vimentin revealed a remarkable disorganisation of the Müller cell orientation in certain parts of the retina from β2^−/−/γ3^−/− mice. B, B′: Immunostaining for GFAP, a marker for reactive gliosis, indicates a strong upregulation in Müller cells of β2^−/−/γ3^−/− mice. In the wild-type retina, retinal astrocytes located in the ganglion cell layer (GCL) express GFAP (arrow in B). In the knockout animal, not only glial elements in the GCL (astrocytes and Müller cell endfeet, arrow in B′) are GFAP-positive, but also Müller cells spanning all retinal layers (B′). C,C′: An immunostaining against utrophin which may connect channels to the DAP complex did not reveal remarkable differences between wild-type and knockout animals. Arrows point to intraretinal blood vessels which are contacted by immunopositive glial membranes. D-D″: The inner limiting membrane could be stained with a pan-laminin antibody at the vitreal side of wholemount preparations from wild-type animals (D). This staining was lacking in some parts of the β2^−/−/γ3^−/− retina (D′). Arrowheads in D′ mark a laminin-positive blood vessel. In regions where the inner limiting membrane still existed, it displayed a netlike structure (D″). E, E′: The aquaporin-4 located at retinal blood vessels was similar in both groups (arrows mark a blood vessel in the outer plexiform layer (OPL). Whereas Müller cell endfeet contacting the inner limiting membrane (arrowheads) were aquaporin-4-positive in the wild-type retina, this staining could not be observed in β2^−/−/γ3^−/− mice. F, F′: Prominent Kir4.1 immunostaining was found in Müller cell endfeet in the GCL. This staining was found to be reduced in the knockout situation. Weak immunostaining was found in structures likely to be Müller cell processes in the inner plexiform layer (IPL, arrows in F). Photoreceptor segments displayed strong autofluorescence (lower edge of the images). G: Western blot analysis of aquaporin-4 (30 kDa), Kir4.1 (41 kDa), and GAPDH (36 kDa) as loading control in retinal homogenates of littermate controls (con, left lanes) and of β2^−/−/γ3^−/−animals (β2^−/−/γ3^−/−, right lanes). Protein expression of both channel proteins is lower in β2^−/−/γ3^−/− animals compared to controls. Scale bar in F′ applies to all panels except D″. Retinal slices in A-C and E-F are shown with the vitreal side upwards. INL, inner nuclear layer, ONL, outer nuclear layer.</p