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Fig. 4 Requirement of CCT activity for BBSome assembly, (A) CCT chaperonins are required for BBSome assembly. HA-BBS9 was cotransfected with siRNAs against CCT1, CCT2, CCT3, or control in HEK293T cells, and BBS9-associated BBSome subunits were probed by immunoprecipitation followed by immunoblotting. (B) Chaperonin-like BBS proteins are required for the interaction between BBS7 and CCT chaperonins. Expression of BBS6, BBS10, and BBS12 was blocked by RNAi in HEK293T cells, and endogenous BBS7 was immunoprecipitated in control, BBS6-, BBS10-, and BBS12-depleted cells. Association of CCT chaperonins with BBS7 was measured by immunoblotting. (C) MO-mediated knockdown of CCT chaperonins results in Kupffer’s vesicle (KV) defects in zebrafish. Representative images of zebrafish embryos and KVs are shown at Left. (Top) Lateral view of a 10-somite stage embryo with the location of the KV circled, dorsal views; (Middle and Bottom) of a normal KV (arrow) and a reduced KV (arrowhead) at the 10-somite stage. Embryos were injected with 15 ng of indicated MOs, except for cct1, for which a 5-ng dose was used because of severe developmental defects and lethality. The percentage of embryos with reduced or absent KVs are represented for each MO treatment. Sample size and P values are in Table S2. **P < 0.01 determined by Fisher’s exact test compared with control MO-injected group. (D) Retrograde melanosome transport is delayed in cct2 morphants and genetic mutants. Melanosome transport is assessed by monitoring melanosome trafficking in the head region of zebrafish (Top). Below are representative images of the boxed area with dispersed melanosomes of dark-adapted zebrafish before epinephrine treatment (Middle) and contracted melanosomes after epinephrine induced transport (Bottom). Rate of melanosome transport to the endpoint (perinuclear distribution) is noted in seconds on y-axis. Data are represented as mean ± SEM. **P < 0.01, determined by one-way ANOVA and Tukey test. (Scale bar, 100 μm.)