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. 2021 Mar 3;4(5):e201900332. doi: 10.26508/lsa.201900332

Figure 6. Kalirin inhibition hinders migration in MNA cells and perturbs cell polarisation and MT structure.

Figure 6.

(A) Relative migration in 2D exclusion assay after treatment with vehicle, kalirin–GEF1 inhibitor#1 (10 μM) and kalirin–GEF1 inhibitor#2 (5 μM) or after KALRN RNAi. Relative cell migration is quantified via normalization of cell density to vehicle- or siRNA-treated control. Graphs represent mean relative difference in migration + SD. (B) Random walk plots and accumulated migration distances in control IMR-32_NM cell after treatment with kalirin–GEF1 inhibitor#1 or kalirin–GEF1 inhibitor#2, and after 48 h of KALRN RNAi (13 and 10 h, 15-min intervals). Mean values + SD are presented. (C) Time-lapse images of IMR-32_NM after kalirin–GEF1 inhibitor#2 treatment. Nuclei and leading processes are indicated. Scale bar 20 μm. (D) |NC-CC| plots in control IMR-32_NM and after treatment with kalirin–GEF1 inhibitor#2, kalirin–GEF1 inhibitor#1 or KALRN siRNA. (E) The percentage of centrosomes located distally in control, kalirin, or RAC1-suppressed IMR-32 cells. Mean values + SD are presented. (F) DAPI staining showing changes in the nucleus shape in IMR-32 after kalirin–GEF1 inhibition. (G) Box plots demonstrating nuclear roundness in IMR-32 cells treated with RAC1- or kalirin–GEF1 inhibitor. Data represent three independent experiments (819, 1,008, 375, and 772 cells). (H) βIII-tubulin staining in control IMR-32 cells and after treatment with kalirin–GEF1 inhibitor#2 or KALRN RNAi. The representative fields were photographed. Scale bar 100 μm. (I) NNC/NCC angle frequency distribution (left) and NUC and noise-corrected NC-CC distances in 0–40° and 140–180° signatures in concatenated tracks from control (25 cells and 866 cells), RAC1-inhibited (30 cells, 563 timepoints), kalirin–GEF1–inhibited (25 cells and 606 timepoints) and KALRN siRNA treated (30 cells and 860 timepoints) IMR-32_NM (right). (J) Correlation plots between cell velocity and NUC footprint in control IMR-32_NM and after treatment with RAC1 inhibitor, kalirin–GEF1 inhibitor#1, kalirin–GEF1 inhibitor#2, or KALRN siRNA. (K) Phase contrast images, random walk plots, and accumulated migration distances of randomly migrating cells treated with vehicle, RAC1 inhibitor (10 μM), kalirin–GEF1 inhibitor#1 (10 μM) or kalirin–GEF1 inhibitor#2 (5 μM) for 48 h in pseudo-3-D (21 h, 90-min intervals). Mean values + SD are presented.