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

Figure EV2. NTRAS operates as splicing‐regulatory lncRNA.

Figure EV2

  1. Relative expression of NTRAS in HUVECs and HeLa cells, determined by RT–qPCR (n = 4 independent biological replicates).
  2. RT–qPCR‐based identification of accessible regions within NTRAS using RNase H‐mediated cleavage of RNA–DNA heteroduplexes (DNA antisense oligonucleotides AS1 to AS5) in HUVEC cell lysate (n = 1). The oligonucleotide used for probe design is highlighted in red.
  3. Scheme illustrating the affinity selection of endogenous NTRAS–protein complexes for RNA and protein analysis.
  4. Sucrose density gradient ultracentrifugation showing the distribution of NTRAS and hnRNPL (protein). The dashed box indicates the fractions with the greatest overlap of both factors (n = 1).
  5. Illustration of the human NTRAS locus. Displayed is NTRAS (GRCh38.p13; RP11‐354k1.1) and RBPmap‐predicted hnRNPL binding motifs, described elsewhere (Smith et al, 2013).
  6. Western blot‐based validation of NTRAS–hnRNPL interaction following antisense affinity selection of NTRAS in nuclear extracts from normoxic and hypoxic HUVECs (n = 4 independent biological replicates).
  7. Expression of NTRAS in control and NTRAS‐silenced HUVECs used for RNA sequencing (n = 4 independent biological replicates).
  8. hnRNPL mRNA levels in control and hnRNPL‐silenced HUVECs used for RNA sequencing (n = 3 independent biological replicates).
  9. RT–PCR‐based analysis of CD55 intron 7 retention following hnRNPL/NTRAS double knockdown in HUVECs (n = 4 independent biological replicates). Representative agarose gel on the right.
  10. rMATs‐based analysis of alternative splicing events upon silencing of lncRNA lncflow2 in HUVECs (n = 4 independent biological replicates) ES: Exon skipping, MXE: Mutually exclusive exons, A5SS: Alternative 5’ splice site, A3SS: Alternative 3’ splice site, RI: Retained intron.
  11. Validation of hnRNPU silencing in HUVECs by RT–qPCR (n = 4 independent biological replicates).
  12. RT–PCR‐based analysis of TJP1 exon 20 inclusion in hnRNPU‐silenced HUVECs (n = 4 independent biological replicates).

Data information: In (A, F–I, K, L), data are represented as mean ± SEM. n.s.: non‐significant, **P < 0.01, ***P < 0.001. (A, F–H, K, and L) two‐tailed unpaired t‐test, and (I) one‐way ANOVA.

Source data are available online for this figure.