*4.4. ITIR-FCS*

Giant unilamellar vesicles of DOPC or ternary lipid mixtures of DOPC/egg-sphingom yelin/cholesterol (Sigma-Aldrich) were prepared in 100 mM sucrose solution by electroswelling using a Nanion Vesicle Prep Pro device (Nanion, Munich, Germany). Vesicle suspensions in 250 μL of ultra-pure water were pipetted onto coverslips. After 20 min, the supported lipid bilayers were formed and the solution was replaced with 250 μL of ultra-pure water. Objective-type TIRF illumination was used to achieve the thinnest excited sample volume, with a high numerical aperture objective (alphaPlan-FLUAR100; Zeiss, Oberkochen, Germany). The data were acquired using a ProEM512 EMCCD camera (Princeton Instruments, Trenton, NJ, USA) with a 3 ms effective exposure time and 20 × 40 pixel acquisition area per measurement (pixel size 0.16 μm). The ImFCS plugin (https://www.dbs.nus.edu.sg/lab/BFL/imfcs\_image\_j\_plugin.html, accessed on 16 May 2022) for ImageJ software was used for data evaluation (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2018, accessed on 16 May 2022). The data analysis was performed as described earlier [44].

#### *4.5. Imaging of di-4-ANEPPDHQ and Calculation of General Polarization (GP)*

*E. coli* BL21 (DE3) (Thermo Fisher Scientific) and B16-F10 mouse melanoma cells (ATCC) were used in these experiments. For inducible protein expression, the *E. coli* BL21 (DE3) cells were induced with IPTG as described earlier in Section 4 (Figure S3A). In the case of the B16-F10 cells, HSPB1 was inserted into the pcDNA 4/TO (Thermo Fisher Scientific) expression vector, and the pcDNA6/TR vector (Thermo Fisher Scientific) was used as the source of the tetracycline repressor. The cells were co-transfected with both vectors using ExGen 500 (Thermo Fisher Scientific). Colonies were selected by the simultaneous addition of zeocin and blasticidine (both from Thermo Fisher Scientific). The expression of HSPB1 was induced by the addition of doxycycline hyclate (Sigma-Aldrich, D9891) to the cell culture media (2 μg/mL) for 24 h before the experiment. The expression of HSPB1 upon

doxycycline induction, heat shock, and membrane fluidizing BA treatment was assessed by Western blot (Figure S3B).

Bacterial and mammalian cells were labeled with the environment-sensitive, membraneincorporating dye, di-4-ANEPPDHQ (Thermo Fisher Scientific), added to the growth medium at final concentrations of 5 μM and 1.5 μM, respectively. Following a 5 min incubation at RT, image acquisition was carried out on a Leica TCS SP5 confocal system. An argon ion laser at 488 nm was used for excitation, and detection ranges of PMTs were set to 500–580 nm and 620–720 nm for the two emission channels, respectively. The di-4-ANEPPDHQ data were typically displayed as pseudo-colored GP images. The GP values were calculated according to the following equation: GP = (*I*\_(500-580) − G *I*\_(620- 750))/(*I*\_(500-580) + G *I*\_(620-750)) using an ImageJ macro. *I* represents the intensity in each pixel in the indicated spectral channel (numbers are in nm) and *G* represents the calibration factor, which compensates for the differences in the efficiency of collection in the two channels. Further image processing of segmenting cells and cell membranes was performed with CellProfiler [45] in combination with ilastik [46]. More than 1000 ROIs of membranes were segmented on at least 5 images per sample. GP values were read out and sorted into classes to calculate distribution. The Kolmogorov–Smirnov test was performed to analyze the equality of GP distributions in sample pairs using the MATLAB software (MathWorks, Natick, MA, USA).

HSPB1 expression in *E. coli* cells was induced with 0.5 μM IPTG (Sigma-Aldrich) 4 h before the measurements. Doxycycline hyclate (Sigma-Aldrich) was used to induce the expression of HSPB1 in B16F10 cells 24 h before the measurement. Both bacterial and mammalian cells were incubated with BA (Sigma-Aldrich) for 15 min prior to the measurement.

**Supplementary Materials:** The following supporting information is available at: https://www.mdpi. com/article/10.3390/ijms23137317/s1. References [41,47] are cited in the Supplementary Materials.

**Author Contributions:** Conceptualization: L.V. and Z.T.; funding acquisition: L.V. and Z.T.; investigation: B.C., I.G., Z.K., B.D., É.K., V.V.-Z., Z.L. and T.P.; writing—original draft preparation: B.C. and I.G.; writing—review and editing: B.C., I.G. and Z.T.; visualization: I.G., B.C. and Z.K.; project administration: Z.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hungarian Basic Research Fund (OTKA ANN 132280), by the Hungarian Academy of Sciences (Lendület Grant LP2017-7/2017), and by the EU's Horizon 2020 Research and Innovation Program under gran<sup>t</sup> agreemen<sup>t</sup> no. 739593.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated and/or analyzed during this study are available from the corresponding authors on request.

**Acknowledgments:** We are truly grateful for the HSPB1 expression plasmids provided by Mathias Gaestel.

**Conflicts of Interest:** The authors declare no competing financial interest.
