Supplementary Materials and Methods

Supplementary Materials and Methods Cell culture HBEC cells (kind gift from J. Minna’s laboratory in Dallas, TX) were cultured in Keratinocyte serum-free

medium

(Keratinocyte-SFM;

Gibco)

supplemented

with

1

%

(vol/vol)

penicillin-streptomycin solution (10,000 units per milliliter penicillin + 10 mg/mL streptomycin; Gibco), 2 mM L-glutamine (Gibco), 20 mM Hepes buffer solution (Gibco), 30 g/mL G418 sulfate (Gibco), 250 ng/mL puromycin dihydrochloride (Gibco), 5 ng/mL prequalified human recombinant epidermal growth factor 1-53 (EGF 1-53; Gibco), and 50 g/mL bovine pituitary extract (BPE; Gibco). Cells were maintained at 37 °C under 5 % CO​2 ​and 95 % relative humidity atmosphere. Time-lapse microscopy HBEC cells were seeded at density of ~250,000 cells/cm​2 on polystyrene bottomed (TPP) 12-well tissue culture plates few hours before image acquisition was started. Time-lapse phase contrast microscopy was performed on Olympus microscope IX71 using 10X magnification with 5 min repetition rate. The microscope equipped with cage incubator (Life Imaging Services, Switzerland) controlling the temperature at 37 °C and stage top incubator (Life Imaging Services) controlling the relative humidity and CO​2 partial pressure (95 %, 5 % respectively). Images were captured with CCD camera (Retiga 4000R, QImaging, CA) providing field of view (FOV) 1500 x 1500 µm​2​. The system was controlled with MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) Particle image velocimetry (PIV) The velocity field was calculated with PIVlab software ​ [1] in MATLAB environment, using single path with 24 x 24 µm​2 interrogation window and 50 % overlap. The missing vectors and vectors with magnitude larger than 5 times STD were replaced with interpolated data. Orientation analysis The orientation field was obtained by computing the local structure tensor with OrientationJ imageJ plugin ​[2]​ using sliding window of 22.7 x 22.7 µm​2

Fig. Supp. 1 : ​Shape anisotropy of HBEC culture ~ 25 hours after the onset of confluency. Three zoomed images of HBECs. The overlaid green rods represent the director field for each case, which is computed with OrientationJ ImageJ plugin. The scale bar is 50 μm.

Fig. Supp. 2 : ​Isolated HBECs​. ​The scale bar is 50 μm.

Fig. Supp. 3 : ​Anisotropy level of different cell cultures and artificial images. The dimensionless anisotropy level obtained with OrientationJ ImageJ plugin as a function of time, using an interrogation window of 22.7 x 22.7 µm​2​. Three different cell types, mouse myoblast cells (C2C12), HBECs and Madin-Darby Canine Kidney (MDCK) cells were analyzed starting from the onset of confluency, t=0 hr. As a reference values, we measured the anisotropy level for irregular array of circles (diam. 20-30 µm) and irregular array of rods (length 20-30 µm) whose orientation obeys an independent uniform distribution centered at 0 degrees relative to the horizontal axis with a width of 180 degrees.

Fig. Supp. 4 : ​Time evolution of vortex statistics of HBEC collective flows. (a) Distribution of the vortex areas (Figure 2C in the main text). (b) Distribution of the mean vorticity per vortex normalized by the inverse square root of the enstrophy (ѡ​v​/Ω​1/2 in the main text) and (c) the normalized mean vorticity per vortex as a function of the vortex area. The time scale is parametrized with the inverse of the square root of the mean enstrophy Ω​-1/2​.

Fig. Supp. 5 : ​Average nematic director field around ​±½ defects. Method to compute the average flows/nematic order near defects : 1.- ​At a given instant of time, the director field is computed through OrientationJ ImageJ plugin. We identify the nematic defects and their orientation by using the methods described in ​ [3]​. Therefore, for each defect in the field of view, we obtain a value of its topological charge and the angle of its orientation with respect to a referential axis. This method enable us to identify reliably the topological charge of about 9 over 10 nematic defects. 2.- ​At a given instant of time, we collect all the defects with the same topological charge and the corresponding flow field in a small window of size 300 x 300 µm​2 centered at the defect core. We align all the defects along the same direction, meaning that we rotate the director field and the corresponding velocity field according to the angle of the defect orientation. Finally, we average the rotated director and velocity fields for all defects with the same topological charge in the field of view. 3.- These two previous steps are repeated for all frames. As a result, we obtain an averaged director and velocity fields near ​± ½ defects at every instant of time. The averaged velocity field near defects is normalized by the instantaneous value of Ω1/2 . Finally, to obtain Fig. 4 in the main text and Fig. Supp. 5, we compute the average over time and experiments.

Movie Supp. 1 : ​Phase contrast​ ​time-lapse of HBEC monolayer upon onset of confluency, ~ 12 hours after cell seeding. The scale bar is 300 μm.

Movie Supp. 2 : ​Normalized vorticity map dynamics upon onset of confluency, ~ 12 hours after cell seeding. The scale bar is 300 μm.

Movie Supp. 3 : ​Long-time dynamics of the defects and the director field upon onset of confluency, ~ 12 hours after cell seeding. The scale bar is 300 μm.

Movie Supp. 4 : ​Short-time dynamics of the defects and the director field ~ 30 hours after seeding of cells, between 20 to 25 hours after onset of confluency. The scale bar is 300 μm.

References: [1] W. Thielicke and E. J. Stamhuis, J. Open Res. Softw.​ 2​, (2014). [2] R. Rezakhaniha, A. Agianniotis, J. T. C. Schrauwen, A. Griffa, D. Sage, C. V. C. Bouten, F. N. van de Vosse, M. Unser, and N. Stergiopulos, Biomechanics and Modeling in Mechanobiology​ 11​, 461 (2012). [3] A. J. Vromans and L. Giomi, Soft Matter​ 12​, 6490 (2016).