Fig 1.
Assessment of cell migration in both 2D and 3D cell culture following treatments.
Epi stands for epinephrine and HC stands for hydrocortisone. (A) 2D scratch assay. A1: Brightfield images of cell migration at different timepoints following treatment. The dark shaded region indicates the scratch area remaining at 24 hours post-treatment, and the lighter central area shows the remaining scratch at 72 hours. Colored lines highlight the scratch borders. (a) Epinephrine 2 μM (b) Epinephrine 200 nM, (c) Control, (d) Hydrocortisone 500 nM, (e) Hydrocortisone 5 μM.A2: Quantification of scratch closure (%) across different treatment groups over time. (a) Cell migration at 24h post-scratch (b) Cell migration at 72h post-scratch. A3: Snapshots of cell migration in Epi 2 µM (panels a-c), hydrocortisone 5 µM (panels d-f), and control (panels g-j) groups at 12-, 24-, and 36-hours post-treatment (Scale bar: 200 µm). Black marks indicate approximately the same area across time points within each treatment group. Selected individual cells are indicated by arrows to facilitate tracking over time. (B) Spheroid-based migration assay (Scale bar: 200 µm). B1: Microscopic observation of spheroid morphology following treatments. The central dark shadow represents spheroid morphology at day 1, while the lower image layer shows spheroids at day 4. Spheroids and cell spreads are outlined in blue for better visibility. (a) Epinephrine 2 μM (b) Epinephrine 200 nM, (c) Control, (d) Hydrocortisone 500 nM, (e) Hydrocortisone 5 μM. B2: Quantification of spheroid-based migration assay. (a) Heatmap illustrating morphological differences in spheroids across treatments on day 4. The color scale represents morphological parameters, with warmer colors indicating larger values (up to 1) and cooler colors representing smaller values (down to −1.5). Area was normalized based on control, with 1 equaling the area of the control spheroid at day 4 and 0 representing the spheroid area at day 0. For other parameters: Circularity where 1 represents a perfect circle; Aspect Ratio where 1 shows least spheroid elongation; Roundness where 1 indicates least shape deformation; Solidity where 1 indicates the most compact spheroid shape with fewer protrusions. Lollipop graphs illustrate area changes in both spheroids (b) and cell spread regions (c). B3: Example images showing the effect of treatment on cell migration in 3D cell culture on day 4. (a) Cells migrate out of the spheroid following Epinephrine 200 nM treatment. (b) Hydrocortisone 5 µM treatment inhibits cell migration out of the spheroid. Statistical significance is indicated by asterisks (* for p < 0.05, ** for p < 0.01, and *** for p < 0.001).
Fig 2.
Bio-AFM analysis of 2D and 3D samples.
(A) Sample Visualization for 2D and 3D Cultures. A1: Example cell surface topography obtained from forward deflection scanning by AFM (Scan area: 100 × 100 µm²). A2: Image of a spheroid positioned under the AFM tip; the red dot indicates the laser reflection point. (B) Average Young’s Modulus (kPa) values across treatment groups, with standard deviations shown. B1: Changes in average Young’s modulus (kPa) observed in 2D cultured cells. B2: Corresponding trend in 3D spheroid cultures. (C) Topographical visualization of sample cells using Z-axis height and deflection data. AFM images were acquired over a 100 × 100 µm² area. C1: (a) 2D height map acquired from Z-axis scan (scan forward direction); (b) 3D reconstructed surface based on Z-axis scan data; (c) Height profile extracted along the scan axis, where the gray line represents raw surface measurements, and the black line shows the mean-fitted curve for smoother interpretation. C2: (a) 2D deflection image illustrating surface features based on cantilever bending; (b) 3D reconstructed deflection surface; (c) Deflection profile along the scan axis, with the gray line indicating raw deflection signals and the black line corresponding to the fitted curve. ‘Epi’ indicates epinephrine, and ‘HC’ indicates hydrocortisone.
Fig 3.
Changes in cell stiffness after treatment in 2D and 3D culture systems.
Probability distribution of Young’s Modulus (kPa) for each treatment. Control cells are shown in orange and treated cells in teal. Fitted normal distribution curves are overlaid. (A) 2D and (B) 3D cell cultures. Panels A1–A4 and B1–B4 correspond to treatments with epinephrine (2 µM and 200 nM) and hydrocortisone (500 nM and 5 µM), respectively. ‘Epi’ indicates epinephrine, and ‘HC’ indicates hydrocortisone.
Fig 4.
Changes in cell stiffness and deformation profiles following treatment in 2D and 3D cell cultures.
(A) Comparison of Young’s Modulus values (kPa) across treatment groups; each dot represents an individual measurement. Statistical significance is indicated (***p < 0.001). (B) Force versus deformation scatter plots, with each treatment group color-coded as indicated.
Fig 5.
FITC intensity distribution upon each treatment.
Violin plots depicting the distribution of vimentin-associated FITC fluorescence intensity in gated cell populations for each treatment group. Higher intensity values indicate increased vimentin expression. Each distribution reflects the heterogeneity of response within treatments. Epi stands for epinephrine and HC stands for hydrocortisone.
Fig 6.
Microscopy and flow cytometry analysis of vimentin expression under different treatment conditions.
Rows 1–5 correspond to treatment groups:1: Epinephrine (2 μM), 2: Epinephrine (200 nM), 3: Control (untreated), 4: Hydrocortisone (500 nM), 5: Hydrocortisone (5 μM). Panels A1–A5: Flow cytometry density plots showing side scatter area (SSC-A) versus forward scatter area (FSC-A) for the ungated cell populations. Cell density is color-coded, with red indicating the highest and dark blue the lowest density. Panels B1–B5: Histograms of FITC fluorescence intensity in gated cell populations. Blue curves represent background fluorescence from unstained controls; orange curves represent cells stained with FITC-conjugated anti-vimentin antibody. Mean fluorescence intensity (MFI) values are shown for the stained populations. Panels C1–C5: Immunofluorescence microscopy images. (a) Fluorescent images: nuclei stained with DAPI (blue), vimentin with FITC (green); (b) Corresponding brightfield images. Scale bars = 100 μm.
Table 1.
Statistical comparison of FITC-A intensity distributions using the Kolmogorov–Smirnov test.