*2.3. Fabrication of Polyacrylamide Hydrogels with Stiffness Gradients*

A polymer solution containing 20% (*v*/*v*) acrylamide monomer (Bio-Rad) and 0.4% (*v*/*v*) N,N′ -methylenebisacrylamide cross-linker (Bio-Rad) was prepared in 1× Dulbecco's phosphate-buffered saline (PBS, Life Technologies, Gaithersburg, MD, USA) without Mg2+ and Ca2+. An amount of 1 mL aliquots were degassed for 5 min, and 4 µL of 10% (*w*/*v*) ammonium persulfate (APS) (Sigma-Aldrich) was added and mixed for 0.5 s; then, 1.2 µL of N,N,N′ ,N′ -tetramethylethylenediamine (TEMED) (Bio-Rad) was quickly added and mixed for 1 s. An amount of 120 µL of this solution was transferred into a glass mold, and a functionalized glass coverslip was placed over the solution at an angle so that no air remained under the coverslip. The solution was allowed to polymerize for 30 min at room temperature, after which the coverslip was removed from the mold. The gels were rinsed and stored in 1<sup>×</sup> PBS without Mg2+ and Ca2+ for subsequent use. For cell adhesion, Sulfosuccinimidyl 6-(4′ -azido-2′ -nitrophenylamino) hexanoate (sulfo-SANPAH, Thermo Fisher Scientific, Waltham, MA, USA) was applied to the PA gel surface for 15 min under 365 nm UV light (2 times). Then, the gel was coated with collagen I (100 µg/mL) overnight at 4 ◦C. Before the cell seeding, PA gels were washed 3 times with PBS. PA hydrogels with this structure have thickness gradient and apparent stiffness gradient [28].

To measure the thickness of the gel, PA gel was stained with Coomassie brilliant blue G-250 for about 10 min. Using confocal microscope (Leica TCS SP8 X, Germany, Wetzlar, Germany), we obtained a series of Z-stack images and reconstructed to measure the thickness of the PA gel.

An atomic force microscope (AFM, MFP-3D, Asylum Research Inc., Santa Barbara, CA, USA) with the colloidal probe cantilever was used for the apparent Young's modulus measurement. The cantilever used in the experiments was a non-tip cantilever (NSC36, MikroMasch, Watsonville, CA, USA) with a spring constant of 2 N/m. The colloidal probe was assembled, as described previously, by adhering a glass sphere of diameter d = 26.3 µm to the front end of the cantilever [29]. To effectively reduce the adhesion between the probe and the PA gel, the surface of the colloidal probe was coated with a thin layer of PLL-g-PEG (SuSoS AG.) [30]. The AFM measurements were carried out in contact mode with 1~8 µm indentation depths. The measured force–distance curves were recorded and fitted with Hertz model F = <sup>4</sup> <sup>3</sup>(1−υ2) ER0.5δ 1.5 to calculate the reduced Young's modulus E [31], where F is the measured force, R is the probe radius, δ is the indentation distance, and υ = 0.33 is the Poisson ratio. Prior to each force measurement, the spring constant of the colloidal probe is calibrated in situ using the thermal power spectral density method [29].

The topography of PA gel surface was visualized using scanning electron microscopy (SEM, Quanta 200 FEG). PA gels were flash frozen and were then lyophilized overnight. A layer of platinum (Pt) was deposited on the gel surface using a turbomolecular pump coater (Q150T, Quorum Technologies, Lewis, UK) prior to observation to enhance the electrical conductivity of the gel.

In each independent experiment, the PA gel was fabricated under the same condition. The size, thickness, and partitioning of the PA were controlled consistently. The reproducibility of the gel fabrication was verified.

#### *2.4. Cell Immunofluorescence*

JAR cells in hydrogel substrates were fixed in 4% (*w*/*v*) paraformaldehyde in PBS for 15 min at room temperature. The hydrogel was washed twice with PBS, permeabilized in 0.1% (*v*/*v*) triton X-100 in PBS for 15 min, and washed twice more with PBS. JAR cells were blocked in 2.5% (*v*/*v*) goat serum in PBS for 2 h at room temperature to prevent non-specific binding. Then, cells were incubated with anti-vinculin antibody (1:200, Abcam,

GR3270283-14, overnight, 4 ◦C) in goat serum. For secondary staining, cells were washed twice with PBS and incubated with goat anti-rabbit IgH H&L (488 nm) antibody (Abcam, 1:500, GR320844-3, 3 h, room temperature). Directly stained cells were incubated with 1:200 DAPI and phalloidin (546 nm) in goat serum solution (2 h, room temperature) and thoroughly washed with PBS. Confocal microscope (Leica TCS SP8 X, Germany, Wetzlar, Germany) was used for acquiring images.
