Figure 2.
Cell spreading regulates levels of GTP and rates of NCT. (A) Representative confocal images of BJ-5ta cells expressing LINuS plated on polyacrylamide hydrogels with rigidities of 1, 22, 46, or 308 kPa. (B–D) Nuclear volume (1 kPa n = 23, 22 kPa n = 22, 46 kPa n = 21, 308 kPa n = 28; two independent replicates), (C) nuclear height (1 kPa n = 23, 22 kPa n = 22, 46 kPa n = 21, 308 kPa n = 28; two independent replicates), or (D) cellular area (1 kPa n = 81, 22 kPa n = 93, 46 kPa n = 93, 308 kPa n = 93; two independent replicates) of BJ-5ta cells plated on polyacrylamide hydrogels of 1, 22, 46, or 308 kPa rigidities. (E) Quantification of GEVAL30 signal (405 nm/488 nm) in BJ-5ta cells expressing GEVAL30 in cells plated on polyacrylamide hydrogels at stiffnesses of 1 (n = 152), 22 (n = 178), 46 (n = 197), or 308 kPa (n = 172). Results are from two independent replicates. (F) Import and export rates of BJ-5ta cells expressing LINuS plated on polyacrylamide hydrogels with rigidities of 1 (n = 52), 22 (n = 72), 46 (n = 64), or 308 kPa (n = 80) across four independent replicates. (G) Representative confocal images of BJ-5ta cells expressing LINuS 2 or 24 h after being plated. (H) Cellular area of BJ-5ta cells that had been plated for 2 (n = 20) or 24 h (n = 20) across two independent replicates. (I) Quantification of GEVAL signal (405 nm/488 nm) in BJ-5ta cells expressing GEVALNull or GEVAL30 2 h after trypsinization and rounded (2 h, GEVALNull n = 67, GEVAL30 n = 73) or 24 h after trypsinization and spread (24 h, GEVALNull n = 82, GEVAL30 n = 118). Results are from two independent replicates. (J) Import and export rates of BJ-5ta cells expressing LINuS either 2 or 24 h after being plated (2 h n = 41, 24 h n = 44; two independent replicates). (K) Cartoon of MCF10A cells grown into a monolayer, then scratch wounded. Cells were either imaged along the scratch wound (scratch; yellow rectangle) as they migrated into the denuded space or ∼600 μm into the monolayer adjacent from the scratch edge (middle; blue rectangle). (L) Quantification of GEVAL signal (405 nm/488 nm) in MCF10A cells expressing GEVALNull or GEVAL30 within the monolayer (middle, GEVALNull n = 147, GEVAL30 n = 138) or edge of scratch (scratch, GEVALNull n = 153, GEVAL30 n = 106) 2 h after scratch. Results are from two independent replicates. (M) Import and export rates of MCF10A cells expressing LINuS within the monolayer (middle, n = 98) or edge of scratch (scratch, n = 72) 2 h after scratch, across three independent replicates. Significance calculated using unpaired t test (H, I, K, and L), one-way ANOVA with Tukey’s post hoc (D–F), or one way-ANOVA with Dunnett’s post hoc (B and C). ns P > 0.05, P***<0.001, P****<0.0001. Scale bars 10 µm.

Cell spreading regulates levels of GTP and rates of NCT. (A) Representative confocal images of BJ-5ta cells expressing LINuS plated on polyacrylamide hydrogels with rigidities of 1, 22, 46, or 308 kPa. (B–D) Nuclear volume (1 kPa n = 23, 22 kPa n = 22, 46 kPa n = 21, 308 kPa n = 28; two independent replicates), (C) nuclear height (1 kPa n = 23, 22 kPa n = 22, 46 kPa n = 21, 308 kPa n = 28; two independent replicates), or (D) cellular area (1 kPa n = 81, 22 kPa n = 93, 46 kPa n = 93, 308 kPa n = 93; two independent replicates) of BJ-5ta cells plated on polyacrylamide hydrogels of 1, 22, 46, or 308 kPa rigidities. (E) Quantification of GEVAL30 signal (405 nm/488 nm) in BJ-5ta cells expressing GEVAL30 in cells plated on polyacrylamide hydrogels at stiffnesses of 1 (n = 152), 22 (n = 178), 46 (n = 197), or 308 kPa (n = 172). Results are from two independent replicates. (F) Import and export rates of BJ-5ta cells expressing LINuS plated on polyacrylamide hydrogels with rigidities of 1 (n = 52), 22 (n = 72), 46 (n = 64), or 308 kPa (n = 80) across four independent replicates. (G) Representative confocal images of BJ-5ta cells expressing LINuS 2 or 24 h after being plated. (H) Cellular area of BJ-5ta cells that had been plated for 2 (n = 20) or 24 h (n = 20) across two independent replicates. (I) Quantification of GEVAL signal (405 nm/488 nm) in BJ-5ta cells expressing GEVALNull or GEVAL30 2 h after trypsinization and rounded (2 h, GEVALNull n = 67, GEVAL30 n = 73) or 24 h after trypsinization and spread (24 h, GEVALNull n = 82, GEVAL30 n = 118). Results are from two independent replicates. (J) Import and export rates of BJ-5ta cells expressing LINuS either 2 or 24 h after being plated (2 h n = 41, 24 h n = 44; two independent replicates). (K) Cartoon of MCF10A cells grown into a monolayer, then scratch wounded. Cells were either imaged along the scratch wound (scratch; yellow rectangle) as they migrated into the denuded space or ∼600 μm into the monolayer adjacent from the scratch edge (middle; blue rectangle). (L) Quantification of GEVAL signal (405 nm/488 nm) in MCF10A cells expressing GEVALNull or GEVAL30 within the monolayer (middle, GEVALNull n = 147, GEVAL30 n = 138) or edge of scratch (scratch, GEVALNull n = 153, GEVAL30 n = 106) 2 h after scratch. Results are from two independent replicates. (M) Import and export rates of MCF10A cells expressing LINuS within the monolayer (middle, n = 98) or edge of scratch (scratch, n = 72) 2 h after scratch, across three independent replicates. Significance calculated using unpaired t test (H, I, K, and L), one-way ANOVA with Tukey’s post hoc (D–F), or one way-ANOVA with Dunnett’s post hoc (B and C). ns P > 0.05, P***<0.001, P****<0.0001. Scale bars 10 µm.

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