Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: The effects of Jasplakinolide, Latruncilin A, Blebbistatin, and GSK-269962 on 2D random migration. ( Left panels ): The proliferation curves of Hs578T cells seeded in 2D, showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each containing 10 technical replicates. ( Right panels ): The random cell migration speed of subconfluent Hs578T cells seeded in 2D and exposed to concentration ranges of inhibitors. The boxes indicate the same maximum tolerated concentrations boxed as in the ( left panels ). Bosutinib was used as a positive control. The mean ± SEM is shown for 2 biological replicates, each containing 4 technical replicates.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: CyB suppresses 2D migration speed and cell stiffness at non-toxic concentrations, without disrupting F-actin. ( A ): The proliferation curves of Hs578T and MV3 cells seeded in 2D, showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each with 10 technical replicates. n.s., non-significant. ( B ): The random cell migration speed of subconfluent Hs578T and MV3 cells seeded in 2D and exposed to concentration ranges of cyB. The boxes indicate the same maximum tolerated concentrations boxed in the left panel. The mean ± SEM is shown for N = 3 biological replicates, each with 8 technical replicates. n.s., non-significant; * p < 0.05. ( C ): F-actin fibers (Phalloidin, green or red) and nuclei (Hoechst, blue) of Hs578T and MV3 cells seeded in 2D in the absence or presence of the indicted cyB concentrations. The black arrows indicate actin aggregates and structures resembling membrane ruffles appearing with increasing inhibitor concentrations and replacing the F-actin stress fibers observed under control conditions (white arrows). Scalebar = 20 µm. ( D ): The stress fiber length (left; indicated in ( C ) with white arrows) and area of membrane ruffles (right; indicated in ( C ) with black arrows). The mean ± SD for one experiment with two replicates is shown. n.s., non-significant; * p < 0.05; ** p < 0.01; *** p < 0.0001. ( E ): A cartoon illustrating the principle of AFS. Acoustic force pushes the bead upwards, thereby stretching the cells, with the amount of stretch inversely related to stiffness. ( F ): AFS measurements for cells exposed for 24 h to a control medium or cyB (741 nM for Hs578T and 247 nM for MV3), normalized to the measurement before exposure. The mean ± SD for one experiment of two, each containing 2 technical replicates, is shown.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Cytochalasins Suppress 3D Migration of ECM-Embedded Tumoroids at Non-Toxic Concentrations

doi: 10.3390/ijms26147021

Figure Lengend Snippet: CyD suppresses 2D migration speed and cell stiffness at non-toxic concentrations, without disrupting F-actin. ( A ): Proliferation curves of Hs578T and MV3 cells seeded in 2D showing confluence after 24 h of exposure to concentration ranges of the indicated inhibitors, normalized to time point 0. The boxes indicate the highest concentrations tolerated without growth suppression. The mean ± SEM is shown for N = 3 biological replicates, each with 10 technical replicates. ( B ): The random cell migration speed of subconfluent Hs578T and MV3 cells seeded in 2D and exposed to concentration ranges of cyD. The boxes indicate the same maximum tolerated concentrations boxed in the left panel. The mean ± SEM is shown for N = 3 biological replicates, each with 8 technical replicates. n.s., non-significant; * p < 0.05; ** p < 0.01. ( C ): F-actin fibers (Phalloidin, green or red) and nuclei (Hoechst, blue) of Hs578T and MV3 cells seeded in 2D in the absence or presence of the indicted cyD concentrations. The black arrows indicate actin aggregates and structures resembling membrane ruffles appearing with increasing inhibitor concentrations and replacing the F-actin stress fibers observed under control conditions (white arrows). Scalebar = 20 µm. ( D ): the Stress fiber length (left; indicated in ( C ) with white arrows) and area of membrane ruffles (right; indicated in ( C ) with black arrows). The mean ± SD for one experiment with two replicates is shown. n.s, non-significant; * p < 0.05; ** p < 0.01. ( E ): AFS measurements for cells exposed for 24 h to a control medium or cyD (41.2 nM for Hs578T and 123 nM for MV3), normalized to the measurement before exposure. The mean ± SD for one experiment of two, each containing 2 technical replicates, is shown.

Article Snippet: To do so, a subconfluent monolayer of Lenti-X 293T cells (Clontech, 632180, Mountain View, CA, USA) was transfected with a mixture of this plasmid and lentiviral helper vectors pMDLg-RRE, pCMV-VSVG, and pRSV-Rev (Addgene, #12251, #8454, and #12253, Watertown, MA, USA) using polyethylenimine (Polysciences, 23966-2, Warrington, PA, USA).

Techniques: Migration, Concentration Assay, Membrane, Control