Fig. 4: SMC #13 does not affect WASp cellular concentration and WASp-dependent cellular function in healthy naïve primary cells. | Nature Communications

Fig. 4: SMC #13 does not affect WASp cellular concentration and WASp-dependent cellular function in healthy naïve primary cells.

From: Targeting the actin nucleation promoting factor WASp provides a therapeutic approach for hematopoietic malignancies

Fig. 4: SMC #13 does not affect WASp cellular concentration and WASp-dependent cellular function in healthy naïve primary cells.

a PBMCs were incubated with 40 µM SMC #13 or vehicle, and were left unstimulated, or stimulated with 1 µg/ml ionomycin and 2.5 ng/ml PMA for 24 h ((P < 0.0001) and (P = 0.0140 for vehicle unstimulated vs SMC#13 stimulated)). The cells were then lysed and analyzed by immunoblot with anti-WASp antibody and anti-GAPDH as a loading control. Densitometric analysis of the bands was performed with ImageJ, and the values are presented below the blots. WASp cellular concentration levels were normalized to GAPDH and compared relative to the unstimulated vehicle-treated cells. Values are shown as mean ± SEM of three independent experiments. The P values were calculated using one-way anova and Tukey’s multiple comparison test. n.s not significant. b XTT proliferation assay was performed following 40 µM SMC #13 incubation and/or stimulation of PBMCs, as described in a. The graph summarizes the mean percent cell proliferation relative to the control (P = 0.0003 for stimulated vehicle vs SMC#13). Values are shown as mean ± SEM of three independent experiments. The P values were calculated using one-way anova and Tukey’s multiple comparison test. n.s not significant. c PBMCs were incubated with 40 µM SMC #13 or control, left unstimulated or stimulated for 24 h, and then seeded on a chambered coverslip precoated with ICAM-1. Cell migration was assessed using Zeiss Observer Z1 inverted microscope. Left panel: Cell tracking analysis of five cells from a representative movie of each group. Each line represents the pathway of a single cell. The graph on the right summarizes the mean cell displacement (Neg Cont. (−): n = 301; SMC #13 (−): n = 255; Neg Cont. (+): n = 301; SMC #13 (+): n = 264) (P < 0.0001 for Stimulated vehicle control vs SMC#13). Values are shown as mean ± SEM of three independent experiments. The P values were calculated using one-way anova and Tukey’s multiple comparison test. n.s not significant.

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