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. 2022 May 8;23(6):e54157. doi: 10.15252/embr.202154157

Figure 3. NTRAS controls TJP1 splicing and endothelial barrier function.

Figure 3

  1. Scheme depicting the in vitro splicing of a TJP1 exon 19–exon 20 splice substrate featuring an intronic splicing silencer (ISS). hnRNPL binding sites are highlighted in red.
  2. In vitro splicing efficiency of the TJP1 splice substrate, comparing mock, NTRAS‐depleted, and NTRAS‐CA16 motif add‐back conditions (n = 7–12 independent biological replicates).
  3. Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐silenced HUVECs (n = 6 independent biological replicates).
  4. Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right.
  5. Co‐precipitation of TJP1 pre‐mRNA in anti‐hnRNPL RIPs, using nuclear lysates from control and NTRAS‐CA16 motif overexpressing cells (n = 5 independent biological replicates). Representative western blot on the right.
  6. RT–PCR‐based analysis of TJP1 exon 20 inclusion upon NTRAS overexpression and overexpression of the NTRAS‐CA16 motif (n = 8 independent biological replicates).
  7. Endothelial resistance of NTRAS‐silenced HUVECs (n = 3–5 independent biological replicates), analyzed by electrical cell‐substrate impedance sensing (ECIS).
  8. Endothelial resistance of hnRNPL‐silenced HUVECs (n = 3 independent biological replicates), analyzed by ECIS.
  9. Analysis of TJP1 exon 20 inclusion by RT–PCR upon transfection of HUVECs with a control SSO or an SSO masking the exon 20–intron 20 boundary (E20 SSO) (n = 9 independent biological replicates). Representative agarose gel on the right. Schematic outline at the top right.
  10. Endothelial resistance of control SSO‐ or E20 SSO‐transfected HUVECs (n = 4 independent biological replicates), analyzed by ECIS.

Data information: In (B–J), data are represented as mean ± SEM. n.s.: non‐significant, *P < 0.05, **P < 0.01. (B, G–J) two‐tailed unpaired t‐test, (C–E) two‐tailed paired t‐test, and (F) one‐way ANOVA.

Source data are available online for this figure.