Figure 7(A) Fgf8 mRNA expression in E4 chicken right limb bud embryo was analysed by whole-mount in situ hybridisation. The two independent samples of control (2-hydroxypropyl-beta-cyclodextrin (HBC), n = 4, upper panel) and thalidomide-treated (1 µg/µl (3.9 mM), n = 7, lower panel) chicken embryos are shown. Data in bar graph were calculated as relative Fgf8 mRNA expressions with Fgf8 mRNA expressions of HBC as 100. Error bars denote ± standard deviation (HBC, n = 3 or thalidomide, n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (**P < 0.01). (B) Limb skeletal stained with Alcian blue. Skeletal patterning of right forelimb in E10 chicken embryos treated with the vehicle for thalidomide, HBC (control, n = 6) or 1 µg/µl (3.9 mM) thalidomide (Tha) (n = 6) were analysed by Alcian blue staining. Data in bar graphs were calculated as relative bone length with bone length of DMSO as 100. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (*P < 0.05 and **P < 0.01). (C) Immunohistochemical staining of PLZF or SALL4 in chicken forelimb bud. In left panels, endogenous PLZF or SALL4 protein expression were detected using forelimb bud section in chicken embryos treated with the vehicle for thalidomide, HBC (n = 4) or 1 µg/µl (3.9 mM) thalidomide (n = 4). In the data in the right bar graphs, relative intensities of IHC signals on forelimb bud sections in chick embryos were calculated as relative signal intensity with signal intensity of HBC as 100. Error bars denote ± standard deviation (PLZF (n = 3) or SALL4 (n = 4)) and P-values were calculated by student's t-test (NS = Not Significant and *P < 0.05). (D) Photographs show E4 chicken embryos treated with the vehicle for thalidomide, HBC (control, n = 6, C2, C3 and C5), or 1 µg/µl (3.9 mM) thalidomide (n=18, (normal; T5, T6 and T16), (weak phenotype; T2, T7 and T17) or (strong phenotype; T3, T8 and T18) corresponding to immunoblot analysis in Fig 7E. Red arrows show treated regions. Data information: Data in bar graphs in (D) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation ((D), n = 3 P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001). (E) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with the vehicle for thalidomide, HBC (n = 6), or 1 µg/µl (3.9 mM) thalidomide (n = 18). Data information: Data in bar graphs in (E) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01). (F) Photographs show E4 chicken embryos treated with HBC (control, n = 10, C1, C3, C5 and C6), 1 µg/µl (3.9 mM) thalidomide (n = 11, T1, T2, T6 and T9) or 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n = 10, H1, H2, H8 and H9) corresponding to immunoblot analysis in Fig 7G. Red arrows show treated regions. Data information Data in bar graphs in (F) were calculated as relative limb bud length with left limb bud length as one. Error bars denote ± standard deviation (F), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, **P < 0.01, and ***P < 0.001). (G) Immunoblot analysis of PLZF or SALL4 from tissue of E4 chicken forelimb bud. Endogenous PLZF or SALL4 protein expression was detected by immunoblot using chicken embryos treated with HBC (n =10), 1 µg/µl (3.9 mM) thalidomide (n =11) and 1 µg/µl (3.6 mM) 5-hydroxythalidomide (5-HT) (n =10). Data information: Data in bar graphs in (G) were calculated as relative GgPLZF or GgSALL4 band intensity with GgPLZF or GgSALL4, respectively. The band intensity of HBC was 100. Error bars denote ± standard deviation ((E), n = 3 or (G), n = 4). P-values were calculated by one-way ANOVA with Tukey's post-hoc test (NS = Not Significant, *P < 0.05, and **P < 0.01).