Nanodrop

The cells treated with HSDP were detached using trypsin and washed with PBS before storage at -80°C until RNA extraction. For this purpose, a minimum of 1 x10⁶ cells per experimental group were processed using the RNeasy Mini kit (#74104; QIAGEN). The mRNA levels were measured with the NanoDrop (DS-11, DeNovix), followed by cDNA synthesis using RT Master Mix (FSQ-201, TOYOBO, Japan) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) involved reaction mixtures containing 10 μL THUNDERBIRDTM Next SYBR® qPCR Mix (QPS-201, TOYOBO, Japan), 1 μL (10 pmol/μL) of forward primer, 1 μL (10 pmol/mL) of reverse primer, 7 μL of Nuclease-Free Water, and 1 μL of cDNA in a PCR plate. The amplification was carried out with the Rotor-Gene Q 6plex System (Rotor-Gene Q, Qiagen, Germany) through 40 cycles: denaturation at 95°C for 15 s, annealing at 62°C for 1 min, and extension at 72°C for 1 min. Each plate had at least three replications. The quantification of each target gene expression was standardized against the endogenous control gene (GAPDH) and a list of the primers is shown in S1 Table in S1 File. Relative expressions were calculated using the formula:
$$\mathrm{R}=2^{‐\left[\mathrm{\Delta }\mathrm{C}\mathrm{t}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}‐\mathrm{\Delta }\mathrm{C}\mathrm{t}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right]}$$

DNA extraction was completed by using the CTAB (Cetrimonium bromide) method. DNA was quality verified by gel electrophoresis and quantified by nano-drop (Denovix, Wilmington, DE, USA) estimation. Prior to the RAPD (rapid amplified polymorphic DNA) analyses, the DNA was standardized to a similar concentration (20 ng/uL). Twenty RAPD primers (Table S1), 10 bases long, were used in combination to obtain a map of the genetic variability within selected clones from the original single clonal source. Standard PCR reagents and reaction conditions were used with a basic annealing temperature of 45 °C and an extension time of 2.0 min. PCR products were visualised and scored for band presence and absence on a 3% high separation Agarose gel (Sigma, UK). Band pattern was examined using GenAlEx 6.5 [78 (link)].

Spike S1-Fc or RBD-Fc proteins of SARS-1, SARS-2, and MERS- CoVs, and soluble ACE2 were produced by transiently transfecting HEK293T cells with recombinant plasmid constructs of pCAGGS-MERS-S1-Fc, pCAGGS-SARS-1-S1-Fc, pCAGGS-SARS-2-S1-Fc, pCAGGS-MERS-RBD-Fc, pCAGGS-SARS-1-RBD-Fc, pCAGGS-SARS-2-RBD-Fc, or pCAGGS-sACE2. Five days post transfection, supernatants were harvested and the cell debris was removed by centrifugation at 1200 rpm for 10 min. The recombinant proteins were purified using Protein A sepharose affinity chromatography (GE Healthcare) following the protocol described elsewhere [22 (link)]. Similarly, soluble ACE2 was purified using Nickel-NTA agarose beads (Qiagen, Cat No. 1018244, Hilden, Germany) and eluted with 200 mM imidazole. The quality and quantity of the protein were measured by Nanodrop (Denovix, Wilmington, DE, USA), SDS-PAGE, and Western blot analysis.

Stably transfected SK-N-AS cells were seeded onto T75 flasks (1x10⁶ cells), then cultured and harvested at 24, 48 and 72 hours. The procedure was repeated twice (at passage 23 and passage 30). Cells were pelleted and RNA was extracted using Maxwell 16 LEV SimplyRNA kit (Promega) according to manufacturer’s protocol. RNA quality assessment was performed by measuring absorbance (A260/280; 1.9–2.1) on Nanodrop (Denovix DS-11 spectrophotometer) and RNA integrity number (RIN) values (>9.0) with High Sensitivity RNA Screen tape on 2200 Tape station (Agilent Technologies). Expression analysis was performed with Clariom S arrays for five constructs (empty vector, MYO5B^WT, p.L587P, p.G1611S, and p.R1641C) at three time points of proliferation (24h, 48h, 72h) in duplicates by Eurofins Genomics (www.eurofinsgenomics.eu). Expression data on the 40 top-ranked differentially expressed genes from all three MYO5B mutants, i.e. genes present as differential expressed top-candidates in at least two out of three time points in at least two mutants, or at least in one time point in all three mutants, are presented in S5 Table. Next, Gene Ontology analysis by GOrilla (http://cbl-gorilla.cs.technion.ac.il [38 (link)]) and gene set enrichment analysis (GSEA, [39 (link),40 (link)]) was performed on the gene lists. For further details on microarray and enrichment analyses see S1 File.