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Fig. 4

ID: ZDB-IMAGE-201012-111
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. 4 <styled-content toggle='no' style='fixed-case'>NCC</styled-content> apoptosis, cell cycle, and differentiation

Homozygous ALX1^165F/165F NCC (blue) showed an increase in sensitivity to apoptosis when compared to control ALX1^165L/165L NCC (black). The data on the left represent the mean percentage of Annexin V‐positive cells, indicative of apoptosis, as determined by FACS analysis, with the data on the right being an example of one such experiment. Apoptosis was induced by immersion in a 55°C water bath for 10 min. Representative experiment for each condition is shown. Data are represented as pooled mean ± SEM of three independent experiments. 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 the basal apoptosis rate: P = 3 × 10⁻¹² between control NCC basal apoptosis and induced apoptosis, and between ALX1^165F/165F NCC basal apoptosis and induced apoptosis. **Significantly different from control NCC (P = 0.0004).

Expression levels of cyclins CCNA2 (blue) and CCND1 (orange) in NCC at passages 2 and 3 of ALX1^165L/165L and ALX1^165F/165F NCC. The RT–qPCR relative expression values were normalized to RPLP0 and GAPDH expression. Data are represented as pooled mean ± SEM of three experiments on three clones from each genotype. *Significantly different from control NCC at passage 2 (P = 0.001 between control and ALX1^165F/165F NCC at passage 2 for CCNA2, P = 0.0052 for CCND1). **Significantly different from control NCC at passage 3 (P = 0.0494 between control and ALX1^165F/165F NCC for CCNA2, P = 0.0008 for CCND1).

Fluorescence activated cell sorting (FACS) experiments showed that control ALX1^165L/165L NCC (green) exhibited increased expression of mesenchymal markers CD90, CD105, and CD73 with culture time (passages 1 through 4), whereas homozygous ALX1^165F/165F NCC (blue) showed a consistent expression of the markers expressed at passage 1 throughout. Further, control ALX1^165L/165L NCC showed a downregulation of CD57 expression with culture time, while ALX1^165F/165F NCC maintained the same level of CD57 across passages. Data are presented as the mean percentage of positive‐stained cells across passage numbers. 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. For CD90, P = 0.0013 at passage 3 and P = 0.0207 at passage 4. For CD105, P = 0.0016 at passage 2, P = 0.00004 at passage 3 and P = 0.0021 at passage 4. For CD73, P = 0.0060 at passage 2, P = 0.00004 at passage 3 and P = 0.0114 at passage 4. For CD57, P = 0.0026 at passage 2, P = 0.000003 at passage 3 and P = 0.000007 at passage 4.

Acknowledgments

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