## Materials and Methods
### Particle Tracking Microrheology  
The measurement setup for passive PTM, see Figure 1a, was developed by the Biophysics Group, Department of Physics, FAU Erlangen. FluoSpheresTM (Thermo Fisher Scientific, Waltham, MA, USA), carboxylate-modified microspheres, orange fluorescent (540 nm/560 nm) with a diameter of 1.0 \(\upmu\)m were used for the measurements. A microsphere diameter of 1 \(\upmu\)m was estimated to be immobilized by the mucin mesh [17,22] to get bulk-rheological properties of the mucus. In order to prevent the microspheres from adhesion to the mucus ingredients, they were coated with polyethylenglycol (Mn 3000, Sigma-Aldrich, St. Louis, MO, USA), according to a procedure proposed by [PERSON] et al. [27]. Of the 1% microsphere solution, 1-3 \(\upmu\)L were mixed with 10-30 \(\upmu\)L of mucus. Variations were caused due to the unsuitability of mucus for pipetting. The mucus-microsphere mixture was sealed between a glass slide and coverslip (gap width: 0.25 mm) by a Gene Frame (Thermo Fisher Scientific). The cover slip was placed onto a CFI Plan Apochromat microscopy objective lens (Nikon, Minato, Japan) with 100\(\times\) magnification. A 1 W laser (532 nm) was used to excite the fluorescent microspheres. Filters, lenses and a pinhole controlled the intensity and focused the laser beam. Both laser beam and fluorescence passed a dichroic mirror. A Guppy Pro F-031B camera (Allied Vision Technologies GmbH, Stadtroda, Germany) was used to record the microsphere movements. The frame rate was 200 frames/s and 2 \(\times\) 2 binning was used. The whole setup was placed on an active optical table to reduce vibrations. The evaluation process of a microsphere is depicted in Figure 1b. The calculation of the viscoelastic properties of a microsphere surrounding the medium is based on the work of [PERSON] et al. [28]. Single fluorescent beads (I) were tracked and the trajectory (II), was evaluated by an in-house software implemented in Python. Beads in close proximity to the surface were neglected due to possible adhesion to the cover slip. Linear trends of the microspheres movements due to thermal drifts were removed from the data. The mean square displacement (MSD, \(\Delta r^{2}(\tau)\)) was calculated for each microsphere (III). The lag-times \(\tau\) are defined periods of time over which the microspheres movement were evaluated. The MSD is approximated at each lag-time \(\tau_{0}\) by a power-law function [26]  
\[\Delta r^{2}(\tau)\approx\Delta r^{2}(\tau)\Big{(}\frac{\tau}{\tau_{0}}\Big{)} ^{\alpha(\tau_{0})} \tag{1}\]  
with \(\alpha\) as the logarithmic slope of the MSD which equals the diffusive exponent. \[\alpha(\tau_{0})=\left.\frac{d(ln\Delta r^{2}(\tau))}{d(ln(\tau))}\right|_{ \tau_{0}} \tag{2}\]  
A microsphere's movement is subdiffusive if 0 \(<\alpha<\) 1, indicating a viscoelastic medium. In purely viscous fluids, \(\alpha=\) 1 and in purely elastic solids, \(\alpha=\) 0. Finally, the viscoelastic properties (IV) were determined by the Fourier transform of the MSD and the generalized Stokes-Einstein equation. In two-dimensional PTM, the complex shear modulus and subsequently the storage (G') and loss modulus (G\") are calculated as follows [26]:  
\[|G^{*}(\omega_{0})|=\left.\frac{dk_{\mathrm{B}}T}{3\pi Rln\Delta r^{2}(\tau) \Gamma[\alpha(\tau_{0})+1]}\right|_{\tau_{0}=1/\omega_{0}} \tag{3}\]  
\[G^{\prime}(\omega)=|G^{*}(\omega)|cos(\pi\alpha(\omega)/2) \tag{4}\]  
Figure 1: Particle tracking microrheology. (**a**) Measurement setup: A laser beam excited the fluorescent beads in the sample, which was placed on a lens with 100\(\times\) magnification. A camera tracked the movement of the fluorescent microspheres. (**b**) Evaluation of the measurement data. (**i**) Fluorescent microspheres in the sample. (II) Trajectory of a microspheres’ movement. (III) Mean square displacement (MSD) of a microsphere over lag-times (\(\tau\)). (IV) Viscoelastic properties, storage (G’) and loss modulus (G”) of the microspheres surrounding medium, calculated of the MSD.