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Fig. 6. hnRNP K MO inhibits protein but not mRNA expression of select targets in vivo. (A) qRT-PCR (SYBR Green) for identified hnRNP K cytoskeletal target (Arp2, tau, XNIF) and non-target (peripherin) RNAs. Ordinate, fold difference relative to loading control (GAPDH RNA). Differences were insignificant among the groups (one-way ANOVA, P>0.1). W.T., uninjected wild type; Con., control MO-injected; Uni. or Bi., unilaterally or bilaterally injected, respectively, with hnRNP K MO. (B) Chemiluminescent western blot and quantified band intensities for Arp2, tau and XNIF (after normalization to GAPDH) relative to uninjected controls. (1) uninjected controls; (2) unilaterally injected, control MO: 95±6%; 102±8%; 96±5%, respectively; (3) unilaterally injected, hnRNP K MO: 54±6%, 53±9%, 52±4%, respectively; (4) bilaterally injected, hnRNP K MO: 18±3%, 12±2%, 15±3%, respectively. (Ca-Ec) Parasagittal, confocal, optical sections of stage 29/30 embryos injected with MO and immunostained for Arp2 (Ca-Cc), tau (Da-Dc), and XNIF (Ea-Ec). Normal expression is seen with control MO (Ca,Da,Ea) but was effectively suppressed by hnRNP K MO (Cb,Db,Eb). Inhibition of Arp2, XNIF and tau expression was effectively restored by co-injection of hnRNP K RNA with the antisense MO (Cc,Dc,Ec). Note that axon outgrowth was also rescued, as seen with tau and XNIF (Dc,Ec, arrowheads). Optical sections for Arp2 are too close to the midline to view axons (Ca-Cc). SC, spinal cord. Ca-Cc contain notochord (NC), whereas Da-Ec contain motor axons and somites. Rostral is left in Da-Dc and right in Ca-Cc,Ea-Ec. Scale bars: 100 μm. |
