Journal: Cell reports

Article Title: Confinement controls the directional cell responses to fluid forces

doi: 10.1016/j.celrep.2024.114692

Figure Lengend Snippet: (A–D) The percentage of cells that reverse their direction in tight confinement under flow conditions (4.8-nN fluid force). (A) 4T1, 4T07, 168FARN, and 67NR breast cancer cells; ≥28 cells per experiment, 4 experiments; * p < 0.05, ** p < 0.01. (B) VCand TSA-treated 67NR breast cancer cells and VC- and methylstat-treated 4T1 breast cancer cells; ≥30 cells per experiment, 3 experiments; ** p < 0.01. (C) shcontrol and sh LMNA HT-1080 cells and sicontrol and si LMNA MDA-MB-231 cells; ≥24 cells per experiment, 3 experiments, ** p < 0.01. (D) HT-1080 cells expressing EGFP-KASH2ext or EGFP-KASH2 and treated with VC or ionomycin; ≥34 cells per experiment, 4 experiments; * p < 0.05, ** p < 0.01. (E) Upstream-to-downstream ratio of MYH9-mCherry signal in shcontrol and sh LMNA HT-1080 cells before and after flow initiation (4.8-nN fluid force). n ≥ 29 cells, 3 experiments, * p < 0.05 sh LMNA relative to the shcontrol. (F) Quantification of Fluo-4 Direct fluorescence (ΔF/F 0 ) in sicontrol and si LMNA HT-1080 cells in tightly confined microchannels following exposure to a 4.8-nN fluid force. n ≥ 20 cells, 3 experiments, ** p < 0.01. (G and H) The percentage of (G) shcontrol and sh LMNA HT-1080 cells treated with VC or ionomycin and (H) sicontrol and si LMNA HT-1080 cells expressing TRPM7-YFP or YFP-C1 that reverse their direction following exposure to a 4.8-nN fluid force. (G) ≥16 cells per experiment, 4 experiments, ** p < 0.01. (H) ≥24 cells per experiment, 3 experiments; ## p < 0.01 relative to sicontrol YFP-C1, $ $ p < 0.01 relative to sicontrol TRPM7-YFP, ** p < 0.01. (I) The percentage of HT-1080 cells expressing TRPM7-YFP or YFP-C1 that reverse their direction in moderate confinement under flow conditions (~5-nN fluid force). ≥10 cells per experiment, 3 experiments, * p < 0.05. (J) Schematic summarizing the mechanisms underlying upstream migration in tight confinement. Actin polymerizes upstream and is necessary for activation of NHE1, which promotes cell swelling and works together with calcium to induce migration toward higher pressure regions. Calcium accumulates downstream, mirroring the distribution pattern of myosin IIA, whose activation increases the proportion of cells moving upstream. A rigid nucleus, tethered to the cytoskeleton via the LINC complex, facilitates upstream migration by allowing cells to establish cell polarity and increase intracellular levels of calcium in response to fluid forces. Statistical tests: paired Student’s t test (I), unpaired Student’s t test (F), and one-way ANOVA (A) and two-way ANOVA (B, C, D, E, G, and H) followed by Tukey’s multiple-comparisons post hoc test. Values represent mean ± SD (E and F) or mean ± SEM (A–D and G–I). See also .

Article Snippet: si LMNA (h) , Santa Cruz Biotechnology , Catalog #: sc-35776.

Techniques: Expressing, Fluorescence, Migration, Activation Assay

Journal: Cell reports

Article Title: Confinement controls the directional cell responses to fluid forces

doi: 10.1016/j.celrep.2024.114692

Figure Lengend Snippet:

Article Snippet: si LMNA (h) , Santa Cruz Biotechnology , Catalog #: sc-35776.

Techniques: Produced, Control, Blocking Assay, Recombinant, Calcium Assay, Expressing, Plasmid Preparation, Software