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Fig. 5 Inhibition of geranylgeranylation branch causes statin-mediated tendon cell expansion. (A) Schematic of Hmgcr pathway with enzymes (blue), inhibitors (red) and morpholinos (red) indicated. (B,C) scxa expression in control DMSO-treated/DMSO-injected embryos at 56 hpf (B, 100%; n=30) is expanded upon atorvastatin treatment and DMSO injection (C, 100%; n=62). (D-F) scxa expression is rescued to wild-type levels in embryos incubated with atorvastatin from 32 to 56 hpf and injected at 28 hpf with mevalonic acid (D, 94% rescued; n=18), farnesyl pyrophosphate (E, 33% rescued; n=72) or geranylgeranyl pyrophosphate (F, 49% rescued; n=43). (G-J) At 60 hpf, craniofacial scxa expression, compared with controls (G, 100% wild type, n=41), is expanded in fntb-pggt1b morphants (H, 73% expanded, n=103) and pggt1b morphants (I, 60% expanded, n=89), and reduced in fntb morphants (J, 29% reduced; 71% normal expression, n=17). (K-M) qPCR analysis of scxa, sox9a, col2a1 and myod1 expression at 56 hpf after incubation from 32 to 56 hpf. (K) Cholesterol biosynthesis inhibition (using RO48-8071) reduced col2a1, but had no effect on scxa, sox9a and myod1 compared with controls. (L) ROCK/Rho inhibition (using Y-27632) reduced col2a1, but had no effect on scxa, sox9a and myod1 compared with controls. (M) Cdc42 GTPase inhibition (using ML-141) reduced scxa and col2a1, but did not affect sox9a and myod1. (N) Rac GTPase inhibition (using EHT-1864) expanded scxa expression at 56 hpf after incubation from 32 to 56 hpf. (O) EHT-1864 increased the quantity of scxa+ cells at 56 hpf after incubation from 32 to 56 hpf. (P) qPCR analysis of scxa, sox9a, col2a1 and myod1 expression at 56 hpf after incubation from 32 to 56 hpf. EHT-1864 increased expression of scxa and sox9a, but did not affect col2a1 and myod1. For qPCR (K-M,P), n=3, head region, Welch's two-tailed t-test. Data are mean±s.d. (O) Red bars indicate mean; points represent individual embryos; Mann–Whitney-Wilcoxon test. N.S., not significant; *P<0.05; **P<0.01. Ventral views (B-J,N), anterior towards the left.