Bovine serum albumin (bsa)

Cell suspensions of SK-N-SH or RD cells were incubated with EV-D68 strains for 1 h at 4°C using an MOI of 1. As a negative control, viruses were heat inactivated at 62°C for 10 min before incubation with the cells. Subsequently, cells were washed with PBS, fixed with 4% PFA for 15 min, and blocked for 30 min with PBS containing 5% normal goat serum (Dako, Denmark). Cells were incubated with rabbit anti-EV-D68 VP1 (20 μg/ml; GeneTex) in 2 mM EDTA (Sigma-Aldrich) and 0.1% BSA (Aurion) fluorescence-activated cell sorting (FACS) buffer for 1 h at 4°C. After washing 3 times, cells were incubated with a secondary goat-anti rabbit IgG conjugated to Alexa Fluor 594 (10 μg/ml; Life Technologies) in FACS buffer. After incubation, cells were washed 3 times in FACS buffer and analyzed using a BD FACS Lyrics flow cytometer (BD Bioscience, USA). The percentage of cells to which virus attached was determined using FlowJo 10 software (Ashland, OR, USA). Experiments were performed at least 3 times, and each experiment was performed in duplicate.

A Leica Ultracut UCT ultramicrotome was used to prepare ultrathin sections (50 nm). They were blocked in 1% BSA (Aurion, Wageningen, the Netherlands) in PBS buffer for 15 min and then incubated in primary antibodies in a 1:10 dilution overnight at 4 °C. Followed by washes in PBS buffer (6 × 5 min) and incubated with the goat antirat secondary antibody conjugated with 10 nm colloidal gold (Sigma Aldrich, Poland) in a 1:50 dilution for 2 h, followed by washing in PBS buffer and distilled water. Negative controls were created by omitting the primary antibody step (Figure S1C). Lead citrate (Microshop, PIK Instruments Sp. z o.o., Piaseczno, Poland) and URANYLess (Microshop, PIK Instruments Sp. z o.o., Piaseczno, Poland) were added as contrasting agents. The cells were visualized using a Jeol JEM 100 SX microscope (JEOL, Tokyo, Japan) at 80 kV in the Department of Cell Biology and Imaging, Institute of Zoology, Jagiellonian University in Kraków or a Hitachi UHR FE-SEM SU 8010 microscope at 25 kV, housed at the University of Silesia in Katowice.

All postembedding steps except for the incubation with primary antibodies were performed at room temperature. For single and double immunolabeling with SNAP47 antibody, sections were first incubated two times for 5 min in 0.1 M PBXT (PBS, 0.001% Triton X-100, 0.001% Tween 20, pH = 7.4), followed by 90 min incubation in PBXT supplemented with 2% bovine serum albumin (BSA, Sigma-Aldrich, Darmstadt, Germany) and 5% normal goat serum (NGS; PAN Biotech) at room temperature. The sections were next incubated with primary antibodies diluted in the same buffer overnight at 4°C in a humid chamber. After rinsing several times with PBXT, the binding of primary antibodies was visualized by incubating with goat anti-rabbit or goat anti-guinea pig secondary antibodies conjugated to either 5 or 10 nm gold particles (British BioCell, International, Wetzlar, Germany) in PBXT supplemented with 0.5% acetylated BSA (Aurion, Wageningen, Netherlands), for 90 min in a humid chamber. Grids were rinsed several times in PBXT, PBS, and finally in water. Ultrathin sections were finally stained with 2% aqueous uranyl acetate (Merck, Darmstadt, Germany) for 2 min, and with lead citrate for 30 s. Sections were examined using a Zeiss TEM-912 equipped with a digital camera (Proscan 2K Slow-Scan CCD-Camera, Zeiss, Oberkochen, Germany). For negative controls, primary antibodies were omitted.

After 4 h of culture on the micropatterns, cells were fixed with 3.7% formaldehyde in PBS, permeabilized with 0.2% Triton X-100 in TBS (50 mM Tris-HCl, 0.15 M NaCl, pH 7.4) and blocked with 2% BSA (Aurion) in TBS. The samples were then incubated with primary antibodies against p-SMAD1/5/8 (Cell Signaling) or vinculin (Sigma Aldrich) and detected with Alexa 647- or Alexa 488-conjugated, isotype-specific, anti-IgG antibodies (Invitrogen). Actin was labeled with phalloidin-TRITC (Sigma) and nuclei were stained with DAPI (Life Technologies).
ALP was stained with fast blue RR salt in a 0.01% (w/v) naphthol AS-MX solution (Sigma Aldrich) according to the manufacturer’s instructions.
Image averaging and p-SMAD1/5/8 quantification were performed using home-made Image J (National Institutes of Health) routines. Briefly, nuclear and cytoplasmic p-SMAD1/5/8 fluorescence intensities were measured over nucleus and cytoplasm areas, respectively, obtained from binarized nucleus and actin images. The relative amount of nuclear p-SMAD1/5/8 was obtained by subtracting the cytoplasmic intensity from the nuclear intensity.
Actin orientation was evaluated with the Directionality plug-in (http://fiji.sc/wiki/index.php/Directionality) in Image J.