Additional file 1 of Dynamic substrate topographies drive actin- and vimentin-mediated nuclear mechanoprotection events in human fibroblasts

Additional file 1: Figures S1–S14. Fig. S1 Surface profile of dynamic hydrogel with 90 µm-wide grooves. Black profile indicates the mean, SD depicted in gray (n = 3). Fig. S2 Representative confocal microscopy images of fibroblast nuclei after 2 or 3 rounds of topographical changes (90 µm-wide grooves) or on flat gels with light exposure (Flat + L). Merge: DAPI-stained nuclei (blue) and acetylation signal (red). AcH3 = histone acetylation. Scale bar = 100 µm. Fig. S3 Representative confocal microscopy images of fibroblast nuclei after 2 or 3 rounds of topographical changes (90 µm-wide grooves) or on flat gels with light exposure (Flat + L). Merge: DAPI-stained nuclei (blue) and methylation signal (red). H3K9me3 = histone trimethylation. Scale bar = 100 µm. Fig. S4 Influence of dynamic topographical changes on YAP/TAZ localization. A) Confocal image of fibroblasts on a stiff hydrogel (E = 350 kPa). Nucleus (blue), F-actin (green), YAP (red), scale bar = 100 μm. B) Fibroblasts on a flat gel, or on a gel with 2 rounds of topographical changes (90 µm-wide pits). Top panel: YAP (gray). Bottom panel: Nucleus (blue), F-actin (green), YAP (red), scale bar = 10 μm. C) Quantification of YAP nuclear localization (Nuc/Cyt ratio) of dynamic conditions for 2 rounds of topographical changes (90 µm-wide pits). Kruskal–Wallis test with a Dunn’s post test, based on n ≥ 29 nuclei, each condition performed in duplo with 2 technical replicates every experimental round. Data are represented as mean ± SD. Fig. S5 Addition of 0.5 µM trichostatin A (TSA) increases AcH3 signal in fibroblasts. A) Representative confocal immunofluorescence microscopy images. Blue: DAPI, red: AcH3. Scale bar is 50 µm. B) Quantification of AcH3 intensity levels, n ≥ 9 cells. Unpaired t-test with p < 0.0001. Fig. S6 Detection of double-stranded DNA breaks (γH2Ax) upon addition of chemical compounds (0.5 µM trichostatin A (TSA), 10 µM JIB-04, or 20 µM etoposide (positive control)) using immunofluorescence microscopy. Blue: DAPI, purple: γH2Ax. Scale bar is 50 µm. Fig. S7 Addition of 10 µM JIB-04 increases H3K9me3 signal in fibroblasts. A) Representative confocal immunofluorescence microscopy images. Blue: DAPI, red: H3K9me3. Scale bar is 50 µm. B) Quantification of H3K9me3 intensity levels, n ≥ 36 cells. Mann–Whitney test with p < 0.0001. Fig. S8 Representative image of vimentin distribution in fibroblasts, cultured on hydrogels with dynamic topographies (2 rounds of 5 h with 90 µm-wide grooves) or flat hydrogels. Blue: nucleus (DAPI), green: F-actin (phalloidin), purple: static, flat regions hydrogel, red: anti-vimentin. Arrows indicate vimentin expression at the cell periphery, while asterisks indicate the distribution of vimentin around the nucleus. Scale bar is 100 µm. Fig. S9 Expression levels of the indicated proteins were determined for fibroblasts (immortalized wildtype, WT, or vimentin knock-out, KO) by western blotting using protein-specific antibodies (3 independent experiments). Fig. S10 Comparison between normal human dermal fibroblasts (nhDF) and immortalized fibroblasts (imm.) used for the generation of Vim−/− cells. A) Representative immunofluorescence images of fibroblasts after 24 h on fibronectin-coated glass. Blue: nucleus, green: F-actin, scale bar is 100 µm. B) Quantification of nuclear parameters: area (i), solidity (ii), and form factor (iii), Mann–Whitney test with n ≥ 93 cells and p = 0.0478. C) Quantification of AcH3 mean intensity levels, Mann–Whitney test with n ≥ 74 cells and p = 0.0001. D) Quantification of H3k9me3 mean intensity levels, Mann–Whitney test with n ≥ 71 cells. Fig. S11 Dynamic topographies affect nucleus morphology and histone modifications in immortalized dermal fibroblasts. A) Quantification of nucleus morphology: nucleus area (i), solidity (ii), and form factor (iii) after 3 × rounds of topographical changes (flat to 90 µm-wide grooves). Data are represented in violin plots, with dashed lines indicating the median and dotted lines indicating the quartiles. + L = flat gel (not photoresponsive) with light exposure (control). Mann–Whitney test with **** p < 0.0001, *** p = 0.0003, * p = 0.0226, n = 176 cells (control) and n = 271 cells (dynamic). B) Percentage of nuclei located on the flat (static) part of the hydrogel. Data are represented in a box-and-whiskers plot showing the median, minimum value, maximum value, and the 25th and 75th percentiles. Unpaired t-test, p = 0.0040, compared to a random distribution of nuclei. n = 4 hydrogels with at least 3 processed field of views per gel and ≥ 24 cells per field of view. C) Quantification of mean histone acetylation (AcH3) intensity levels in response to dynamic topographies or flat + light (+ L) control conditions; a.u. arbitrary units. n ≥ 88 cells per condition. D) Quantification of mean histone trimethylation (H3K9me3) intensity levels in response to dynamic topographies. n ≥ 88 cells per condition. Mann–Whitney test with ** p = 0.0029, ns, not significant, p > 0.05. Fig. S12 Presence of histone modifications in Vim−/− fibroblasts on dynamic topographies (2 rounds, 90 µm grooves). Blue: DAPI, red: AcH3 or H3K9me3. Scale bar is 50 µm. Fig. S13 Addition of 0.2 µM cytochalasin D (CytD) increases AcH3 signal in fibroblasts after 2 rounds of topographical changes, as detected using confocal immunofluorescence microscopy. Vehicle is DMSO. Blue: DAPI, red: AcH3. Scale bar is 50 µm. Fig. S14 Addition of 0.2 µM cytochalasin D (CytD) increases H3K9me3 signal in fibroblasts after 2 rounds of topographical changes, as detected using confocal immunofluorescence microscopy. Vehicle is DMSO. Blue: DAPI, red: H3K9me3. Scale bar is 50 µm.