DEV02195 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. RNAi experiments of cogc-3 and cogc-1 regions. Schematic diagrams of predicted transcripts in cogc-3 and cogc-1 genomic regions are shown. The data are from WormBase except for those of the longest forms of cogc-3 and cogc-1 transcripts analyzed in our laboratory. The regions corresponding to the sequenced cDNA clones are shown by blue bars. yk863b10 was attached with the trans-splice leader SL2 and spans from +1 to +2548 followed by a poly-A sequence. yk1037h06 is also attached with SL2 and spans from +1 to +953. yk1037h06 spans from +224 to +2436 followed by a poly-A sequence. L1 soaking RNAi was performed in wild-type hermaphrodites. RNAi of the cogc-3 region using N-terminal dsRNA corresponding to the 1st to 4th exons of cogc-3 resulted in DTC migration defects. RNAi of the cogc-1 region using dsRNAs for N-terminal first to 6th exons or C-terminal 10th to 15th exons of cogc-1 resulted in DTC migration defects. RNAi of Y54E10A.16a.2 caused no DTC migration defects. YES or NO stands for a positive or negative result, respectively. These results suggest that the longest mRNAs from these genomic regions are important for correct DTC migration. The following primers were used to PCR amplify the templates for in vitro synthesis of dsRNAs. N-terminal T7-cogc-3 primers for 1-654 nucleotides: 5¢-GTAATACGACTCACTATAGGGCATGGAAATAGATCATATGCGAAC-3¢ and 5¢-GTAATACGACTCACTATAGGGCCTCCAACTGAGCCTGTCTATC-3¢. N-terminal T7-cogc-1 primers for 1-905 nucleotides: 5¢-GTAATACGACTCACTATAGGGCATGGACGTTGACCGGCTCATGC-3¢ and 5¢-GTAATACGACTCACTATAGGGCTTTTCAATCCACGCAGCGCATTTCTCG-3¢. C-terminal T7-cogc-1 primers for 1566-2364 nucleotides: 5¢-GTAATACGACTCACTATAGGGCAGATTTCTGCGAAGTCGTTGTCG-3¢  and 5¢-GTAATACGACTCACTATAGGGCTTAATTATTAGCTCCTCCGAACCAGCTGGAAG-3¢.

• Supplemental Figure 2 -

  Fig. S2. MIG-23-GFP localization in L2 larvae. Confocal images of MIG-23-GFP in body wall muscle cells in wild-type (A), cogc-3(k181) (B) and cogc-1(k179) (C) L2 larvae. MIG-23-GFP was expressed in a punctate pattern in the muscle cells of wild-type, cogc-3(k181) and cogc-1(k179) animals. The boundaries of the muscle cells are depicted by broken white lines. Scale bar: 10 mm.

• Supplemental Figure 3 -

  Fig. S3. PNA-lectin blot analysis. Mixed-staged worm lysates (20 mg/lane) were subjected to electrophoresis through a 5-10% gradient SDS polyacrylamide gel and proteins were blotted onto a nitrocellulose filter. After blocking with 3% BSA in PBST, the filter was incubated with HRP-PNA (1 mg/ml, Honen) at room temperature for 1 hour and bands were detected by ECL chemiluminescence. In samples from cogc-3 and cogc-1 mutants, protein bands at 25, 42, 77, 155 and >220 kDa had higher intensity compared with those bands in the wild-type sample (large arrowheads). The 55 and 33 kDa bands (small arrowheads) were nearly equivalent in samples from wild-type, cogc-3, cogc-1 animals. PNA bound more strongly to immature glycans than mature glycans, suggesting that glycosylation of multiple proteins is abnormal in cogc-3 and cogc-1 mutants. a-Tubulin was used as a loading control.