Yang X , Li J , Zeng W , Li C , Mao B .

             cell migration
             cell motility

	Figure S1. Elp3 interacts with xSlug and xSlug rescues neural crest migration in the Elp3 morphants. (a) mElp3 pulls down xSlug in co-IP experiment when co-expressed in HEK 293 cells. WCL, whole cell lysate. IB, immunoblot. IP, immunoprecipitation. (b) Co-expression of xSlug mRNA (150 pg/cell at 4-cell stage) in Xenopus embryo restored neural crest migration in Elp3 morphants. Whole-mount in situ hybridization with probes for neural crest markers was processed at St.19-21. LacZ mRNA was co-injected to trace the injected sides (stained red on the left sides). (c) Percentages of embryos with reduced migration of neural crest as shown in (b). The results are from three independent experiments (error bars represent SDs).
	Figure 1. Expression of the Elp3 during early Xenopus development.(a) RT-PCR analysis of Elp3 expression at different stages (St.0 to St.39). (b–f). Whole-mount in situ hybridization of xElp3. Elp3 transcript is detected in the animal pole at St.6.5 (b, lateral view, animal pole to the top). At St.15, Elp3 is expressed at the anterior neural plate and its border (c, dorsal view, anterior to the bottom), and at the late neurulation, Elp3 is most abundant in cranial neural crest (d, frontal view, dorsal to the top). At tailbud stage, Elp3 is mainly expressed in the branchial arches (BA) and eyes (e,f).
	Figure 2. Knockdown of Elp3 inhibits cranial neural crest migration in Xenopus.(a–f) Elp3 MO (25 ng) with or without Elp3 mRNA (0.2 ng) was injected into one cell of four-cell-stage embryos, and whole-mount in situ hybridization with probes for neural crest markers was processed at St.19–21. LacZ mRNA was co-injected to trace the injected sides (stained red on the right sides). (g) Percentages of embryos with reduced migration of neural crest as shown in (a–f). The results are from three independent experiments (error bars represent SDs).
	Figure 3. Elp3 is required for cranial neural crest (CNC) migration in explants and transplantation experiments.(a) CNC explants were dissected from early neurula embryos, plated on FN (10 μg/ml), and imaged at 0, 5, 12, and 20 h. The surface areas of each explant at every image point were measured, and the ratios of the areas to the values at 0 hour are shown by the line plot (b). The data were represented as the means ± standard deviation (SD). **P < 0.01; ***P < 0.001, two-tailed T-test. (c) GFP mRNA (100 pg) was injected into one cell at the 4-cell stage with or without Elp3 morpholino (25 ng). At St.15–16, GFP-labeled CNC explants were dissected and grafted into normal host embryos. The status of the fluorescent neural crest was imaged at the tailbud stage (St.35).
	Figure 4. Elp3 interacts with and stabilizes Snail1 through inhibiting its ubiquitination.(a,b) Co-IP assays showing the interaction between Snail1 and Elp3. (c) Schematic representation of the Snail1 deletion constructs. (d) In co-IP experiments, Elp3 pulled down full length and ∆SNAG Snail1 efficiently, but not ∆Znf Snail1. (e) Co-expression of Elp3 stabilizes Snail1 in 293 cells with or without MG132 treatment (10 μM). The cells were lysed 48 hours after transfection and processed for Western blot analysis. MG132 was added 6–8 hours prior to harvesting. (f) Co-expression of mElp3 stabilizes mSnail1 in Xenopus embryo. The injected embryos were lysed at stage 9 and processed for Western blot analysis. (g,h) Elp3 affects the stability of Snail1. HEK293 cells were transiently transfected with the indicated plasmids. At 48 hours post transfection, cycloheximide (CHX) was added to all of the samples, and the cells were then harvested at the timepoints indicated (0 h, 2 h, 4 h, and 6 h). The levels of Snail1 were determined by Western blotting using the anti-Myc antibody (g). In all cases, α-tubulin was used as a loading control. The relative levels of Snail1 were quantified densitometrically and normalized against α-tubulin (h). The data shown in (h) were the average of three independent experiments and represented as the means ± standard error of mean (SEM). (i) Elp3 inhibits Snail1 ubiquitination. HEK293 cells were transiently transfected using the indicated plasmids and were treated for 4–5 hours with MG132 before harvesting. Snail1 proteins were immunoprecipitated using Strep-Tactin beads and were probed with an anti-ubiquitin antibody. (j) Co-IP experiments showing that Elp3 does not interfere with the Snail1/β-Trcp interaction. HEK293 cells were transiently transfected with the indicated plasmids and then were treated for 6–8 hours with or without MG132 before harvesting. The proteins were immunoprecipitated with anti-Flag M2 beads and then were detected with the antibody against different tags. WCL, whole cell lysate. IB, immunoblot. IP, immunoprecipitation. CHX, cycloheximide.
	Figure 5. Snail1 or N-cadherin rescues neural crest migration in the Elp3 morphants.(a–f) Co-injection with mSnail1 mRNA (0.5 ng) rescues the migration of neural crest cells in the Elp3 morphants. (g) Percentages of embryos with reduced migration of neural crest as shown in (a–f). The results are from three independent experiments (error bars represent SDs). (h,i) Knockdown of Elp3 by morpholino down-regulates the N-cadherin level, while co-injection with Elp3 mRNA (0.2 ng) restores its expression. (j) Percentages of embryos with reduced expression of N-cadherin as shown in (h,i). The results are from three independent experiments (error bars represent SDs). (k) The expression levels of N-cadherin in embryos injected with Elp3 morpholino and Elp3 mRNA at St 13. Both cells of the 2-cell stage embryos were injected with Elp3 morpholino (12.5 ng/cell) alone, together with rescue mRNA (0.1 ng/cell), or Elp3 mRNA (0.5 ng/cell) alone. Total RNAs from whole embryos were subjected to RT-PCR analysis for the expression of the indicated genes (cycle numbers: ODC, 24; N-cadherin, 32). (l) Elp3 is required for the expression of N-cadherin in induced animal caps. The embryos were injected animally at 2-cell stage with tBR (0.2 ng/embryo) and Wnt7b (0.5 ng/embryo) to induce neural crest fate. Elp3 morpholino and/or mRNA were co-injected as indicated. Animal caps were dissected at stage 9 and harvested when control embryos reached St 19–21. Total RNAs from the animal caps were subjected to RT-PCR analysis for the expression of the indicated genes (cycle numbers: histone H4, 24; N-cadherin, 31). (m–p) Co-injection with mN-cadherin mRNA (0.1 ng) rescues the migration of neural crest cells. LacZ mRNA was co-injected to trace the injected sides (stained red on the right sides). (q) Percentages of embryos with reduced migration of neural crest as shown in (m–p). The results are from three independent experiments (error bars represent SDs).

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