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TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

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Stiffness and stiffness response to force decrease during TGF-β induced EMT. (A, B) NMuMG cells were treated with 100 pM TGF-β for 48 h to induce EMT. Histogram of NMuMG cell stiffness for (A) epithelial state (untreated) and (B) mesenchymal-state. Insets, fluorescence images are cells stained with phalloidin to show actin structures. (C) Average cell stiffness of NMuMG cells for untreated (n = 90) and TGF-β treated (n = 98) populations. **p < 0.001. (D) Average stiffness response for untreated (n = 30) and TGF-β treated (n = 25) populations. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. (E) PANC-1 cells were treated with 300 ng/mL BMP-2 for 72 h to induce EMT. Average PANC-1 cell stiffness for untreated (n = 86) and BMP-2 treated cells (n = 61). *Stiffness difference relative to untreated cells at the p < 0.05 level. (F) Average PANC-1 stiffness response for untreated (n = 20) and BMP-2 treated (n = 15) cells. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. Error bars represent SEM; data were collected from three independent experiments.
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Figure 2: Stiffness and stiffness response to force decrease during TGF-β induced EMT. (A, B) NMuMG cells were treated with 100 pM TGF-β for 48 h to induce EMT. Histogram of NMuMG cell stiffness for (A) epithelial state (untreated) and (B) mesenchymal-state. Insets, fluorescence images are cells stained with phalloidin to show actin structures. (C) Average cell stiffness of NMuMG cells for untreated (n = 90) and TGF-β treated (n = 98) populations. **p < 0.001. (D) Average stiffness response for untreated (n = 30) and TGF-β treated (n = 25) populations. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. (E) PANC-1 cells were treated with 300 ng/mL BMP-2 for 72 h to induce EMT. Average PANC-1 cell stiffness for untreated (n = 86) and BMP-2 treated cells (n = 61). *Stiffness difference relative to untreated cells at the p < 0.05 level. (F) Average PANC-1 stiffness response for untreated (n = 20) and BMP-2 treated (n = 15) cells. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. Error bars represent SEM; data were collected from three independent experiments.

Mentions: To determine the effect of EMT on cell stiffness and stiffening response, we induced EMT in normal murine mammary gland (NMuMG) epithelial cells, a well-established TGF-β–induced EMT model (Piek et al., 1999; Yu et al., 2002; Xie et al., 2004). A magnetic tweezers system (Fisher et al., 2006; O'Brien et al., 2008) was then used to apply force via integrins (Matthews et al., 2006; Guilluy et al., 2011) to the cytoskeleton through externally attached paramagnetic beads coated with fibronectin (FN). The viscoelastic response of a cell was observed by monitoring the displacement of a bound bead over time during force application (Figure 1A). To quantify the mechanical phenotype in terms of stiffness and stiffening response, we calculated the time-dependent compliance of the cell and fitted it to a Jeffrey model for viscoelastic liquids (Figure 1B; Larson 1999). The spring constant obtained during the first pulse of force provided a measure of stiffness, and by normalizing the spring constants of subsequent force pulses to the first, we obtained the stiffness-response to force, or stiffening response. Two classifications of mechanical response were observed: a stiffening response (Figure 1C) and a softening response (Figure 1D). TGF-β–induced EMT was verified by monitoring reduced E-cadherin levels (see later discussion of Figure 4A) and actin reorganization (insets, Figure 2, A and B).


TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.

Osborne LD, Li GZ, How T, O'Brien ET, Blobe GC, Superfine R, Mythreye K - Mol. Biol. Cell (2014)

Stiffness and stiffness response to force decrease during TGF-β induced EMT. (A, B) NMuMG cells were treated with 100 pM TGF-β for 48 h to induce EMT. Histogram of NMuMG cell stiffness for (A) epithelial state (untreated) and (B) mesenchymal-state. Insets, fluorescence images are cells stained with phalloidin to show actin structures. (C) Average cell stiffness of NMuMG cells for untreated (n = 90) and TGF-β treated (n = 98) populations. **p < 0.001. (D) Average stiffness response for untreated (n = 30) and TGF-β treated (n = 25) populations. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. (E) PANC-1 cells were treated with 300 ng/mL BMP-2 for 72 h to induce EMT. Average PANC-1 cell stiffness for untreated (n = 86) and BMP-2 treated cells (n = 61). *Stiffness difference relative to untreated cells at the p < 0.05 level. (F) Average PANC-1 stiffness response for untreated (n = 20) and BMP-2 treated (n = 15) cells. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. Error bars represent SEM; data were collected from three independent experiments.
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Figure 2: Stiffness and stiffness response to force decrease during TGF-β induced EMT. (A, B) NMuMG cells were treated with 100 pM TGF-β for 48 h to induce EMT. Histogram of NMuMG cell stiffness for (A) epithelial state (untreated) and (B) mesenchymal-state. Insets, fluorescence images are cells stained with phalloidin to show actin structures. (C) Average cell stiffness of NMuMG cells for untreated (n = 90) and TGF-β treated (n = 98) populations. **p < 0.001. (D) Average stiffness response for untreated (n = 30) and TGF-β treated (n = 25) populations. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. (E) PANC-1 cells were treated with 300 ng/mL BMP-2 for 72 h to induce EMT. Average PANC-1 cell stiffness for untreated (n = 86) and BMP-2 treated cells (n = 61). *Stiffness difference relative to untreated cells at the p < 0.05 level. (F) Average PANC-1 stiffness response for untreated (n = 20) and BMP-2 treated (n = 15) cells. #Stiffness difference of Gx from G1 at the p < 0.05 level. *Stiffness response (Gx/G1) difference between conditions at the p < 0.05 level. Error bars represent SEM; data were collected from three independent experiments.
Mentions: To determine the effect of EMT on cell stiffness and stiffening response, we induced EMT in normal murine mammary gland (NMuMG) epithelial cells, a well-established TGF-β–induced EMT model (Piek et al., 1999; Yu et al., 2002; Xie et al., 2004). A magnetic tweezers system (Fisher et al., 2006; O'Brien et al., 2008) was then used to apply force via integrins (Matthews et al., 2006; Guilluy et al., 2011) to the cytoskeleton through externally attached paramagnetic beads coated with fibronectin (FN). The viscoelastic response of a cell was observed by monitoring the displacement of a bound bead over time during force application (Figure 1A). To quantify the mechanical phenotype in terms of stiffness and stiffening response, we calculated the time-dependent compliance of the cell and fitted it to a Jeffrey model for viscoelastic liquids (Figure 1B; Larson 1999). The spring constant obtained during the first pulse of force provided a measure of stiffness, and by normalizing the spring constants of subsequent force pulses to the first, we obtained the stiffness-response to force, or stiffening response. Two classifications of mechanical response were observed: a stiffening response (Figure 1C) and a softening response (Figure 1D). TGF-β–induced EMT was verified by monitoring reduced E-cadherin levels (see later discussion of Figure 4A) and actin reorganization (insets, Figure 2, A and B).

Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

Show MeSH
Related in: MedlinePlus