Figure 3. Transcription increases with the extent of chromatin stretching.

(a) A schematic of the process of Fluorescence in situ Hybridization (FISH) to quantify force-induced transcription at the location of transgene insertions with Cy3-conjugated mixed-probes (see Methods) to detect the expression of DHFR mRNA. Arrows represent stretching stress on the chromatin. (b) From the left, a brightfield image of a live CHO cell (its nucleus was highlighted with dashed lines) with a magnetic bead (black dot) (the white arrow represents the direction of the bead center displacement); middle left, GFP-LacI spots (green) were shown indicating the location of the transgene insertions; middle right, DHFR mRNA expression was quantified using Cy3-conjugated FISH probes (red); right, overlay of Cy3-FISH (red) with GFP-LacI (green) showing very close vicinity of the two. Stress was applied at 17.5 Pa at 0.3 Hz for 1 hour. Scale bar, 5 μm. (c) Summarized data of DHFR expression at transgene insertions as a function of the stress angles. Each cell was stressed only at one particular angle (this is the case for all FISH experiments). Controls (No Stress) were the cells in the same culture dish without attached magnetic beads so that they were exposed to the same magnetic field but no mechanical stress. The stress was applied for 1 hr at 0.3 Hz. Mean ± s.e.m.; n>50 cells per condition; *** P<0.001. (d) Gene upregulation depends on stress amplitudes. Ctrl, 0: cells in the same dish as those bound with beads (for plotting simplicity, all control cells data were lumped together). RGD, 0: cells bound with Arg-Gly-Asp peptides coated beads but no stress. PLL, 8.8 or PLL, 17.5: cells bound with poly-L-lysine coated magnetic beads (one bead per cell), applied with a 8.8-Pa or a 17.5-Pa stress. PLL+DMSO, 17.5: cells bound with PLL-beads, pretreated with 0.1% Dimethyl sulfoxide (DMSO) (a solvent for Latrunculin A (LatA)), applied with a 17.5-Pa stress. PLL+LatA, 17.5: cells bound with PLL-beads, pretreated with 1 μM LatA for 30 min, applied with a 17.5-Pa stress. RGD, 8.8 or RGD, 17.5: cells bound with RGD coated beads, applied with either an 8.8-Pa or a 17.5-Pa stress. All stresses were applied at 0.3 Hz for 1 hr. Mean ± s.e.m.; n=60 cells for Ctr, 0; 25 cells for RGD, 0; 33 cells for PLL, 8.8; 41 cells for PLL, 17.5; 19 cells for PLL+DMSO, 17.5; 26 cells for PLL+DMSO, 17.5; 43 cells for RGD, 8.8; and 31 for RGD, 17.5; 4 independent experiments. (e) Gene upregulation by stress depends on duration of stress application (17.5 Pa at 0.3 Hz). DHFR transcription increased in cells stressed via RGD-coated beads relative to controls. Mean ± s.e.m.; without stress: n=31, 32, and 30 cells at 15, 30, and 60 min, respectively; with stress: n=47, 30, and 31 cells at 15, 30, and 60 min, respectively. * P<0.05; *** P<0.001. Note that stress angles were lumped together in (d) and (e) for each condition.