Cellmask stain

FISH was performed with ViewRNA ISH Cell Assay kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following Affymetrix probe sets were used: Net1 cat# VB1-3034209, Cyb5r3 Cat# VB1-18647, Cenpb cat# VB1-18648, mouse Rab13 cat# VB1-14374, human Rab13 cat# VA1-12225, Pkp4 Cat# VB4-600264, Kif18b cat# VA6-3170686, Ddr2 cat# VB1-14375, Arpc3 cat# VB1-14507, P4hb cat# VB6-15898. To detect polyA RNAs, LNA modified oligodT probes (30 nucleotides) labeled with ATTO 655 were added during hybridization, pre-amplification, amplification and last hybridization steps of ViewRNA ISH Cell Assay. Cell mask stain (Thermo Fisher Scientific) was used to identify the cell outlines. Samples were mounted with ProLong Gold antifade reagent (Thermo scientific).

For in situ hybridization, cells were plated on fibronectin-coated polyacrylamide gels, or on fibronectin-coated 20 μm micropatterned line tracks (CYTOOchips Motility, CYTOO), or on fibronectin-coated glass coverslips for 2–3 hours and subsequently fixed in 4% paraformaldehyde for ten minutes. FISH was performed with QuantiGene ViewRNA ISH Cell Assay kit (Affymetrix, cat# QVCM0001) according to the manufacturer’s instructions. The Affymetrix probe sets used were: Ddr2 cat# VB1-14375, RhoA cat# VB6-14572, Cyb5r3 VB1-18647, Arpc3 cat# VB6-14571. To detect PolyA RNAs, LNA modified oligodT probes (30 nucleotides) labeled with ATTO-655 were added during hybridization, pre-amplification, amplification and last hybridization steps of QuantiGene ViewRNA ISH Cell Assay. Nuclei were stained using DAPI and cells were additionally stained with Cell mask stain (Thermo Fisher Scientific) to obtain cell outlines.

Cells (10⁵) from overnight cultures of wild-type (WT), mutant, and complement strains were washed and resuspended in phosphate-buffered saline (PBS; Gibco), mixed with 200 nM DiOC₆ (Life Technologies, Inc.) stain or 100× CellMask stain (Thermo Fisher) for membrane staining. Cells were incubated in 96-well black-bottomed Greiner plates, and readings for relative fluorescence units (RFU) over 30 min of incubation were taken by a spectrophotometric plate reader (SpectraMax M5; Molecular Devices, CA) with excitation at 482 nm and emission at 504 nm for DiOC₆ and excitation at 485 nm and emission at 535 nm for CellMask green live stain.