Carboxyfluorescein

For scrape-loading to assess the permeability of gap junctional channels to miRNA, a fluorescence-tagged miRNA (miR-F), which is constructed by a 25 nt miRNA (5′-CCT CTT ACC TCA GTT ACA ATT TATA-3′) labeled with carboxyfluorescein on its 3′ end (Gene Tools, Inc. OR), was used as we previously reported28 (link). This miR-F was proven to be not hybridized or degraded and also had no fluorescent tag removal in the cytoplasm38 39 . For performance of scrape-loading, cells were grown to confluence and incubated in 100 μM miR-F. Parallel lines were cut by a razor blade. After 30 min, cells were washed with HBSS and the diffusion of miR-F was imaged. The distance from the scrape edge to the point where the average fluorescence intensity dropped to 1.5X the background intensity were measured by NIH imageJ software (NIH, Bethesda, MD, USA).

Antisense morpholino oligonucleotides (MOs) either non-modified or conjugated to carboxyfluorescein were purchased from GeneTools. Xenopus laevis Vps25 splice blocking MOs: 5′-GCTGTGCCCACCACACCGCTCACGT-3′ and 5′-CAACTTTCCTGGAACACAGTATGTT-3′. Control MO: 5′-CCTCTTACCTCAGTTACAATTTATA 3′.