Fig. 2

Eribulin normalizes CAF traits in the CDM system. A Illustration depicting the experimental conditions for CAF-generated CDMs treated with DMSO (blue) or eribulin (red) during matrix production. B Representative images of CAF-generated CDMs that resulted from the assorted experimental conditions used during the 5-day matrix production. Images show fibronectin fibers detected by indirect immunofluorescence and digitally pseudo-colored to depict the orientation angles of ECM fibers. The colored bar on the right represents the angle distributions and was normalized to show mode angle as 0° (cyan). The scale bar in the bottom right image corresponds to 50 μm. Note that CAFs treated with paclitaxel at 3 nM were not quantified due to the lack of fibrillogenesis as indicated by the yellow arrows pointing to ECM gaps and by the low cellularity and ECM thickness, measured below 7 μm, as shown in accompanying Supplemental Fig. 1 A. The graph indicates the levels of fiber alignment, quantified as the percentage of fibers oriented within 15° from the mode angle. C Representative cropped immunoblots (for full matching blots see Supplemental Fig. 2) of lysates collected at the end of CAF-generated CDM production showing representative bands for palladin, phospho-SMAD2/3 (pSMAD2/3), and total SMAD2/3 (SMAD), using GAPDH as an intracellular protein loading control. Accompanying graphs depict levels of palladin and activated SMAD (pSMAD2/3) gauged from immunoblots and normalized to levels attained under DMSO treated conditions. D The total intensities of phospho-FAK (pFAK) and activated α5β1-integrin, calculated using the MetaMorph software 7.8.0.0 and measuring total intensities with the same inclusive intensity thresholds set for all conditions, were normalized to cell numbers (nuclei counts). Experiments were repeated 4 times while a minimum of 6 images were acquired per sample. For statistical significance, one-way ANOVA was used to compare to DMSO vehicle. * P < 0.05; ** p < 0.01; *** P < 0.001; **** p < 0.0001