Fig. S2

Larval physical appearance and PED-6 expression in small molecule inhibitor treated larvae. (A-E) Bright field images of control DMSO- (A), olomoucine- (B), IPA-3- (C), LimKi- (D) and cucurbitacin E- (E) treated larvae at 5 dpf. All small molecule inhibitors were treated from 3 to 5 dpf. Lateral views, anterior to the left. These small molecule inhibitors did not induce significant change in larval physical appearance at the concentrations used. Note that none of IPA3-treated larvae (C) showed blood circulation defects (n>50). (F-G) The same concentration of IPA-3 (1uM) treatment from 5 to 48 hours induced a phenotype that is indistinguishable from that of Pak1 moprholino injected embryos (Kelly et al., 2014). As seen in Pak1 morpholino injected embryos, these IPA-3-treated embryos did not have blood circulation (n = 9 out of 11). These data suggest that IPA-3 is inhibiting Pak1 function, and Pak1 is dispensable from proper cardiac morphogenesis after 3 pdf. (H-J) Fluorescent images of DMSO- and olomoucine-treated wild-type larvae soaked in PED6. PED6 is processed by phospholipase and fluorescence was detected in the intestine in all treated larvae. However, in some olomoucine-treated larvae, PED6 fluorescence in the gallbladder became weaker (I) or absent (J), compared to strong fluorescence (H) in wild-type larvae. White arrows point to the gallbladder. (K) Percentage of larvae showing high, low and no PED6 fluorescence in the gallbladder of DMSO-, olomoucine-, IPA-3-, LimKi- and cucurbitacin E- treated wild-type larvae at 5 dpf.