Fig 2: These are diagrams of our cell culture set up. (A) Represents the TransEpithelial Electrical Resistance (TEER) well set up. We had two 24 well plates with transwell inserts. The wells which had transwell inserts are represented with a blue outline. (B) Represents the chamber slide set up. These were Lab-TeK II Chamber Slides with 4 wells. Cells were grown in the chambers outlined in pink. ZO-1 (Thermo Fisher 339100) and Claudin-1 (Abcam ab15098) were used as primary antibodies with a secondary stain of donkey anti mouse IgG 488 (Thermo Fisher A10042) and donkey anti rabbit 594 (Thermo Fisher A21202). Mouse IgG1 Isotype Control (Thermo Fisher MA5-14453) was used as a negative control.

Fig 3: SH-SY5Y cells differentiated with B-27 show no significant difference in neurite length compared with SH-SY5Y cells differentiated with BDNF. (A) SH-SY5Y cells were differentiated for 5 days with DMEM/F-12 + 10 µM RA, in conjunction with varying combinations of neurite outgrowth-stimulating molecules (B-27 or BDNF) or serum starvation (0.5% FBS). Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1:500 mouse anti-ß(III)-Tubulin (R&D Systems, #MAB1195) and visualized with 1:400 Alexa Fluor® 488-conjugated donkey anti-mouse (Thermo Scientific, #A21202) (green). Nuclei were counterstained with 1:5,000 DAPI (blue). Images taken at ×20 magnification. Scale bar = 100 µm. (B) Longest neurite length and total neurite length measurements were taken using the FIJI software (Schindelin et al., 2012). Three independent experimental replicates were performed and 80 cells per condition were measured in total. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com. ns = not significant. BDNF, brain-derived neurotrophic factor; DAPI, 4',6-Diamidino-2-phenylindole; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PFA, Paraformaldehyde; RA, retinoic acid.

Fig 4: Expression of GEF-H1 in response to wild-type and mutant forms of MKL1. Representative images of megakaryocyte progenitors transduced with MKL1 deletion constructs, cultured in thrombopoietin-only medium, and immunostained for a-tubulin and GEF-H1. Tubulin was detected with mouse anti-tubulin (1:250, ThermoFisher A11126) and AlexaFluor 488-labeled donkey anti-mouse AlexaFluor 488 (1:500, LifeTechnologies A21202). GEF-H1 was detected with rabbit anti-GEF-H1 (1:50, AbCam ab155785) and AlexaFluor 555-labeled donkey anti-rabbit (1:500, LifeTechnologies A31572). Hoechst 33342 was used to identify nuclei.