MOESM1 of The choroid plexus may be an underestimated site of tumor invasion to the brain: an in vitro study using neuroblastoma cell lines

Additional file 1: Figure 1. Effect of a pretreatment of the HIBCPP layer with the actin microfilament-disrupting agent Cytochalasin D on the transmigration rate of IMR32 and SH-SY5Y neuroblastoma cell lines (a) and barrier integrity, as assessed by TEER (b) and dextran flux (c) measurements. Transmigration experiments were performed and transmigration rates were determined as described in Materials and Methods section (a). Before transmigration experiments, filters with HIBCPPs were incubated for 75 min with 1 µg.ml−1 Cytochalasin D (Sigma) diluted in serum-free medium containing 0.5 % BSA (‘+ cytochalasin D’ condition). In parallel, control filters were incubated with serum-free medium containing 0.5 % BSA (‘- cytochalasin D’ condition). The TEER was measured before the treatment and after the treatment to confirm break-down of the barrier properties (b, ‘before’ and ‘cyto D’ conditions). All filters were then placed in new wells containing medium without Cytochalasin D, the transmigration experiment was launched. 5 µl Dextran-TexasRed (MW: 3000 Da, Life Technologies) were added to the upper compartment of the inserts together with IMR32 or SH-SY5Y cells, in order to monitor permeability of HIBCPPs treated with and without cytochalasin D during the experiment (c). After 4 h of transmigration, the TEER was measured again (b, condition ‘after’). TEER values increased again and the experiment was stopped. The fluid in the lower compartments was collected for determination of the amount of Dextran having crossed the barrier during the experiment by fluorescence measurement using a Tecan 200 M Infinite Multiwell reader (c). All results were expressed as mean ± SD from two independent experiments, each performed in triplicates. Statistical significance was assessed by unpaired t-tests. A p-value < 0.05 was considered as significant. Statistical analyses were performed using GraphPad Prism 5.0 for Windows (GraphPad Software, San Diego, California, USA)

Additional file 1: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Measurement of HIBCPP cell viability after infection with E-30 with the Live/Dead and lactate dehydrogenase (LDH) assay. (A) Live/dead assay on HIBCPP cells after 28 h of infection with E-30 Bastianni, 13-311, 13-759, and 14-397. Representative images of four independent experiments each performed in triplicates are shown. (B) LDH activity assay on HIBCPP cells after 28 h of infection. LDH-release into the supernatant by HIBCPP cells was measured after 28 h of infection with different echovirus strains. Data are shown as mean + SD of four independent experiments each performed in triplicates. Technical triplicates were used during the analytical evaluation. (TIFF 1907 kb

Additional file 11: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

E-30 sequence alignments. Positions identical to those of Bastianni are indicated as dots. (A) Amino acid alignment of the P1 region. The VP4, VP3, VP2, and VP1 protein sequences are shown in red, green, blue, and purple, respectively. (B) Amino acid alignment of the P2 region. The protein 2C, 2B, and 2A sequences are shown in raspberry, orange, and light blue, respectively. (C) Amino acid alignment of the P3 region. The 3C protease, VPg, and RNA-dependent RNA polymerase sequences are shown in green, purple, and red, respectively. (D) Nucleotide alignment of 5′UTR regions. (E) Nucleotide alignment of 3′UTR regions. (PDF 3120 kb

Additional file 12: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Amino acid substitutions observed between E-30 Bast. and the outbreak strains. To illustrate differences in between the E-30 strains used, a table was designed with the data that has already been displayed in Additional file 11. The positions that matched between 13-311 and the other three E-30 strains are highlighted in green; those that were different are left blank (white). 13-311 and 14-397 vary in 10 amino acids, whereas 13-311 and 13-759 vary in 70 amino acids. (PDF 139 kb

Additional file 9: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Despite longer incubation periods, 13-759 and 14-397 do not show an impact on barrier integrity. Barrier integrity of HIBCPP cells was evaluated via measurement of the transepithelial electrical resistance (TEER) (A) at indicated time points after infection with E-30 Bastianni, 13-311, 13-759, or 14-397. TEER values at the start of the experiment (white bars), after 24 h (light gray) and after 48 h (dark gray) are shown.Data are shown as mean + SD of 2 independent experiments carried out in quadruples (B) Live/dead assay on HIBCPP cells after 48 h of infection with E-30 Bastianni, 13-311, 13-759, and 14-397. Representative images of two independent experiments each performed in triplicates are shown (C) HIBCPP cells were infected with E-30 Bastianni, 13-311, 13-759, and 14-397 for 48 h and ZO1 staining was compared; cell layers were stained for nuclei with DAPI (shown here in blue), VP1 (shown here in green), and ZO-1 (red). For detailed description of image acquisition and preparation, please refer to Fig. 2. Two images per strain showing different grouping of parallel staining are displayed horizontally (column one: only ZO-1; column two: DAPI, VP-1, and ZO-1; E-30 strains are listed vertically. The images shown are representative examples of multiple stainings taken from two independent experiments each performed in duplicates. (TIFF 13334 kb

Additional file 10: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Verification of virulence after E-30 passage across the HIBCPP cells. HIBCPP cells were infected with E-30 Bastianni, 13-311, 13-759, and 14-397 for 24 and 48 h. (A) Shows the viral genome copies (shown in copies/ml) harvested after 24 or 48 h from the lower compartment (apical cell side). A schematic representation of the experimental setup indicates the experimental procedure. The undiluted supernatant was added to confluent RD monolayers, and the cytopathic effect was observed over 24 (B) and 48 h (C). Virulence was confirmed through the RD cells detaching from the well, rounding off and finally lysing. All viral strains show to have a cytopathic effect on RD cells. The images are representative frames from 2 experiments. (TIFF 9646 kb

Additional file 3: of Strain-dependent effects of clinical echovirus 30 outbreak isolates at the blood-CSF barrier

Paracellular PMN migration across HIBCPP in FIB/SEM-inverted images. FIB/SEM serial sections of paracellular polymorphonuclear neutrophil (PMN) migration through HIBCPP cells. Shows inverted SEM images from the condition HIBCPP+PMN + T-cells+E-30 + IL8. The video shows a paracellular migrating PMN presented in Fig. 8a, b in orthoslices. (AVI 12638 kb