ZFIN Image: Costa et al., 2020, Figure 3

Image

Figure Caption

Figure 3 CRISPR‐Cas9 excision of the 3′UTR localisation element of <italic>RAB13</italic> disrupts mRNA targeting

CRISPR‐Cas9 strategy to derive HUVECs with an excision of the LE in the RAB13 3′UTR (∆LE) and parallel generation of wild‐type (Wt) control cells. The Wt RAB13 exon 8 is represented with its coding sequence in dark and the 3′UTR in clear boxes. The 5′ and 3′ gRNA‐targeted regions are represented with green lines. Arrows: relative positions of the forward (f) and reverse (r) PCR primers used to identify HUVECs with CRISPR‐Cas9-mediated excision of the LE.

Representative genotyping PCR demonstrates the band size shift in ∆LE HUVECs.

Detailed DNA sequence depicting nucleotide positions within the RAB13 3′UTR of Wt and ∆LE HUVECs.

Wt and ∆LE HUVEC RNAseq mapped reads depicting RAB13 exon usage.

Quantification of RAB13 mRNA smFISH spot number in Wt and ∆LE HUVECs (n = 3 experiments; ns: not significant; unpaired t test).

Number of RAB13 mRNA smFISH spots plotted against the respective Polarisation Index (PI) (n = 29 cells; ns: not significant; linear regression).

Left: representative Western blotting (WB) of Wt and ∆LE HUVECs. Right: densitometry analysis of WB data (n = 3 samples; ns: not significant; unpaired t test).

smFISH co‐detection of RAB13 and control GAPDH in Wt and ∆LE motile HUVECs cultured under subconfluent conditions.

PI of RAB13 and GAPDH co‐detected in Wt and ∆LE HUVECs (n = 29 cells; ***P < 0.001, ns: not significant; Mann–Whitney test).

RAB13 PI plotted against respective GAPDH PI. The slope of the coloured lines represents the average RAB13/GAPDH PI ratio; the dashed grey line represents a 1:1 ratio (n = 29 cells).

Data information: 3 Wt and 3 ∆LE HUVECs independent clones were used to collect data (E–J). Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 20 μm (H). Bar charts are presented as means ± s.d.Source data are available online for this figure.

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ EMBO J.