Figure 1: Fibroblasts reciprocally balance cellular contractility and microtubule acetylation.

(a,c) Representative western blots of cell lysates of HFFs treated with the indicated reagents. (b) Quantification of a and c. Relative levels of acetylated microtubules or phosphorylated MLC, each normalized against GAPDH (n>3 experiments per condition). (d) Representative confocal images of HFFs cultured in 3D collagen then treated as indicated, or HFFs transiently expressing GFP-tagged wild-type tubulin (control, WT-MT), GFP-tagged acetyl-mimetic mutant of tubulin (α-tubulin K40Q; HyperAcMT) or GFP-tagged acetyl-null tubulin mutant (α-tubulin K40A; HypoAcMT). F-actin and collagen fibres were visualized by rhodamine-phalloidin (green) or confocal reflection microscopy (red), respectively. All images were acquired using identical instrument settings. (e) Quantification of reflection intensity by collagen fibres in d, expressed as intensity compared with the control. Ten cells were counted for each drug treatment study, and more than 70 cells were counted for each experiment (n>3). (f) Examples of force traction maps of HFFs stably expressing different tubulin mutants. HypoAcMT (K40R) represents another type of acetyl-null tubulin mutation that allows maintenance of the overall shape and positive charge of the K-40 residue. The colours and numbers indicate local traction force in Pascals; cell outlines are indicated by purple lines. (g) Quantification of average traction force magnitude per cell in Pascals (n>8). Data are shown as mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001. Student’s t-test was used for b, and two-way analysis of variance (ANOVA) followed by Tukey post test was used for e and g. (d) Scale bar, 50 μm; (f) scale bar, 20 μm.