Originpro 2018b

All the data were analyzed by analysis of variance using the Origin Pro 2018b (OriginLab Corporation., Northampton, Massachusetts, USA) statistics program, and the means were compared by the least significant difference test (LSD) at significance levels of 0.05 and 0.01.

The toxicities of the compounds were determined by measuring the median effective concentration (EC₅₀) for each compound. This was done using Vibrio Fischeri bacteria and a Microtox Model 500 analyser (Modern Water plc, Guildford, UK) according to the manufacturer’s protocol. The compounds under investigation and the bacteria were incubated for 5 min and 15 min in order to determine an EC₅₀ value for both time points. Based on the data, the EC₅₀ values were determined by the software-based fitting using the MicrotoxOmni 4.2 software. Typically, four differing concentration points were enough to determine the EC₅₀ value. However, for certain compounds, namely [TMGH][OMs], [DBNH][OMs] and [TEAH][OMs], the fitting performed by the MicrotoxOmni software did not pass our quality standards (R² > 0.95). Therefore, a higher number of concentration points were measured, and fitting was completed using ‘Origin’ (OriginPro 2018b, OriginLab Corporation, Northampton, MA, USA). The fitting was performed using the Dose-Response curve with variable Hill slope -function of the software. The optimisation algorithm was Levenberg Marquardt. An example fit for data measured for [TEAH][OMs] is shown in Fig. S2 in Supplementary Information.

Thermogravimetric (TG) and differential thermogravimetric (DTG) analyses were performed under a nitrogen atmosphere using a TGA Q500 thermal gravimetric analyzer (TA Instruments, New Castle, DE, USA). The nitrogen flow into the furnace was maintained at a rate of 90 mL·min^–1. A small square was cut from the paper samples weighing approximately 4–6 mg, which was placed into a platinum crucible. The samples were heated from approximately 30 °C to a target temperature of 600 °C with a heating rate of 10 °C min⁻¹. The obtained thermogravimetric traces for TG and DTG were analyzed with Universal Analysis 2000 software (TA Instruments). Each sample analysis was performed in triplicate. The curves were averaged, and the resulting curves were plotted with OriginPro 2018b software (OriginLab Corp., Northampton, MA, USA).

Statistical evaluation using Pearson correlations, one-way and two-way analysis of variance (ANOVA) with Tukey’s post hoc test at a level of significance of p < 0.05, and principal component analysis (PCA) was performed with OriginPro 2018b (OriginLab, Northampton, MA, USA). Heritability was calculated as $h^2=1-\frac{\vartheta }{{\sigma }_G^2}$ , where $\vartheta$ is the mean variance of a difference of two best linear unbiased predictors (BLUP) and ${\sigma }_G^2$ the genetic variance [16 (link),17 (link)].

Three models of diffusion in yeast were tested on the collected data: a two-compartmental (Model 0 when fitted in the full range of b-values, Model 0B when fitted in a given interval of b-values), a two-compartmental with an intercept (Model 1) and a three-compartmental model (Model 2). All of the models were fitted to the data in OriginPro2018b software. The overall formula for the echo attenuation in the sample for a particular t_m is: $\frac{E}{E_0} = \left(e^{-\frac{2\tau }{T_2}}·e^{-\frac{t_m}{T_1}}\right)·{{\displaystyle \sum }}_{i=1}^n{p}_i·e^{-\left({\gamma }^2{G}^2{\tau }^2\left(t_m + \frac{2}{3}\tau \right)\right)D_i} +y_0,$
where T₁ and T₂ relaxation times were taken from the distributions shown in Figure 1, i is i-th compartment in the sample, n is the number of compartments, E is the echo amplitude for the given τ and t_m and E₀ is the echo amplitude for the minimal τ and t_m [13 (link)], p_i is the molar fraction of i-th population and ${{\displaystyle \sum }}_{i=1}^n{p}_i=1$ , D_i (m²s⁻¹) is the apparent diffusion coefficient in the i-th compartment, and: ${\left(\gamma G\tau \right)}^2\left(t_m+\frac{2}{3}\tau \right) = b,$
which is a diffusion weighting factor in units (sm⁻²). y₀ is the intercept and is equal to zero for Model 0, Model 0B and Model 2.

To ensure stable pH values, an imaging buffer (2× SSC, 50 mM Tris⋅HCl pH 8, 10% (wt/vol) glucose, 2 mM Trolox (Sigma-Aldrich), 0.5 mg/mL glucose oxidase (Sigma-Aldrich), 40 μg/mL catalase (Sigma-Aldrich)) was used for a maximum of 2 h after the addition of enzymes. LatticeSIM z-stacks were acquired with Elyra7 using a C-apochromat 63×/1.2 water immersion objective and 642 nm diode laser (150 mW, 5% laser power, 50 ms exposure time, 15 phases, 196 nm slicing, z-range ~10 µm) for excitation of Cy5 and Hoechst34580 was excited by the 405 nm diode laser. Detection and quantification of FISH clusters were performed on maximum-intensity projections with an ImageJ macro [29 (link)].
The lowest automatic threshold value used for the analysis of SARS-CoV-2-infected cells was also applied to non-infected control cells labeled by the FISH probes. Using this threshold (1100, 65,535), no single particle was detected in the maximum-intensity projections of control cells. Analysis of normal distribution, statistical significance, and graphical illustration of parameters particle area, circularity, and particle density was performed with OriginPro 2018b.