Coreldraw x7

Cuticle thickness was measured to evaluate the effects of this property on the laser signal response. For this, the previously dissected cuticle samples were cut transversally lengthwise down the midpoint of the sample. Samples were then placed on an aluminium scanning electron microscope (SEM) stub using carbon tape. Digital images were captured and analysed with an FEI Inspect S50 microscope (FEI, Hillsboro, OR, USA). Measurements were made with the graphics software Coreldraw X7 (Corel Corporation, Ottawa, Canada) using the dimension tool and adjusting the scale to real-world values using the scale bar from each individual SEM image (electronic supplementary material, figure S1).

Fluorescence microscopy was performed basically as described before [20 (link)]. Cells were grown statically at 37 °C in C + Y medium, and the expression of the GFP fusion proteins was induced by adding desired concentration of ZnSO₄. To stain the unfixed cells with fluorescently labelled vancomycin (VanFL) (Molecular Probes) the pneumococcal cultures were grown to OD₆₀₀ 0.2 in C + Y medium, and the samples were labelled with 0.1 μg ml⁻¹ of Van-FL/vancomycin (50:50) mixture for 5 min at 37 °C before examination. A quantity of 2 μl of the culture was spotted onto a microscope slide and covered with a 1 % PBS agarose slab. The samples were observed using an Olympus Cell^R IX 81 microscope equipped with an Olympus FV2T Digital B/W Fireware Camera and 100× oil immersion objective (N.A. 1.3) (phase contrast). The images were modified for publication using Cell^R Version 2.0 software, ImageJ (http://rsb.info.nih.gov/ij/) and CorelDRAW X7 (Corel Corporation). Fluorescence intensity line scans were acquired using ImageJ and plotted as a function of cell length measured with MicrobeTracker Suite [53 (link)].

HEK293 cells transfected with a combination of human wild-type or mutant α1β3γ2 cDNAs (ratio 1:1:5) were fixed with fresh 4% PFA followed by 2x washing with 1x PBS. Cells were then incubated for 1 h at RT in blocking solution (10% normal donkey serum (NDS), 5% BSA, 0.3% Triton X-100/PBS). After blocking, the cells were treated with 0.65 µg/ml primary antibody in buffer (5% NDS, 2% BSA, 0.3% Triton X-100/PBS) for 16 h at 4 °C^{45 (link)}. After washing the cells 3 × 20 min in PBS, immunoreactivities were revealed using anti-rabbit (1:300) secondary antibodies (Jackson ImmunoResearch) conjugated with Cy3 fluorophores diluted in 2% BSA in PBS. Nuclei were stained with Hoechst. After the final washes (3 × 40 min) the coverslips with the stained cells were mounted on microscopy glass slides with glycerol gelatine. Imaging was executed on a Zeiss 780LSM laser-scanning microscope and assembled in CorelDraw X7 (Corel Corporation).

FoxG1/slp1 sequence alignment was conducted using Jalview Version 2.9 [44 (link)]. Statistical analyses were conducted using GraphPad Prism 6.0 or R Statistical Software. Non parametric statistics were used in the event a data set did not meet the assumptions of normality or equal variance between groups. Graphical representations were prepared using GraphPad Prism 6.07 and CorelDraw X7.

Data were analyzed with OriginPro 2017 (OriginLab, Northampton, MA, USA) and figures were edited with CorelDRAW X7 (Corel Corporation, Ottawa, ON, Canada). Data are represented as mean ± SEM of at least three independent biological replicates/experiments. Significant differences of two independent values were evaluated by unpaired Student’s t-test. Whereas one-way ANOVA followed by Tukey’s post hoc test was used when groups of data were compared with each other. The type of statistical test is indicated in the figure legends. In case no specific test is mentioned, ANOVA was performed.