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Fig. 5

ID: ZDB-IMAGE-201012-113
Publication: Pini et al., 2020 - ALX1-related frontonasal dysplasia results from defective neural crest cell development and migration
All Figures: Figures for Pini et al., 2020

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Fig. 5 <italic><styled-content toggle='no' style='fixed-case'>ALX</styled-content>1</italic><sup><italic>165F/165F</italic></sup><styled-content toggle='no' style='fixed-case'>NCC</styled-content> show a migration defect and a difference in <styled-content toggle='no' style='fixed-case'>BMP</styled-content> secretion

Mutant ALX1^165F/165F NCC (blue) exhibited a migration defect in timed coverage of the central clearing of the wound assay when compared with control ALX1^165L/165L NCC (black). Data are presented as percent area recovery of the central circular clear area of the wound assay by migrating NCC at the end of a 24‐h period. For fluorescent pictures, cells were stained in serum‐free media containing 3.6 μM CellTracker Green CMFDA (Life Technologies) for 30 min at 37°C and allowed to recover for 30 min before starting the experiment. Images were acquired every 6 h using a Keyence BZ‐X800 microscope. Surface area analyses and percentages of coverage were measured using ImageJ software (NIH). The NCC were monitored over 24 h. The data are represented as the average of the percentage of closure ± SEM. Scale bar = 200 μm. Data obtained of each clone from three independent experiments were pooled, and the mathematical mean was calculated. SEM was used to determine the standard error. To test statistical significance, an ANOVA test was performed. A P‐value < 0.05 was considered to be statistically significant. *Significantly different from control NCC (P < 0.0001).

Multiplex analysis of BMP2 and BMP9 in the supernatant of cultured NCC showed that ALX1^165F/165F NCC (blue) secrete less BMP2 and more BMP9 compared to control ALX1^165L/165L NCC (green). Data are represented as pooled mean ± SEM of three clones from each genotype. Statistical significance was determined with an ANOVA test. A P‐value < 0.05 was considered significant. *Significantly different from control NCC (P = 0.0424 for BMP2 and P = 0.0192 for BMP9).

Addition of soluble BMP2 or CV2, a BMP9 antagonist, to the culture medium could partially rescue the migration defect of ALX1^165F/165F NCC. At the beginning of the assay, 10, 50, or 100 ng/ml of soluble BMP2, CV2, or a combination of the two at 100 ng/ml each were added to the culture medium, and the cells were monitored over the next 24 h. The data are represented as the average of the percentage of closure ± SEM. Scale bar: 400 μm.

Acknowledgments

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