Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 3. QKI-6 is implicated in HDAC7 signalling and promotes its splicing. (A,B) QKI-6 was overexpressed by plasmid transfection on day 1 of SMC differentiation and cells were harvested 48 h later. QKI-6 overexpression was shown to significantly induce expression of HDAC7 along with members of the downstream SMC signalling pathway (SRF and myocardin) at the mRNA and protein level. (C) Knockdown of QKI on day 3 of differentiation for 72 h suppressed SRF expression significantly, as shown by qRT- PCR. (D) Levels of HDAC7 and its splicing were observed throughout miPS-SMC differentiation. HDAC7 splicing increased, showing a significant increase in expression at day 6 of differentiation in comparison to splicing at day 2 miPS- SMCs as revealed by RT-qPCR. Levels of total HDAC7 were not significantly changed. (E) miPS-SMCs between day 2 and day 4 of differentiation were screened for the induction of a range of relevant splicing factors, including QKI-5 and QKI-6. qRT-PCR revealed only QKI-6 was significantly induced at day 4 of differentiation in comparison to day 2 differentiated cells. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Plasmid Preparation, Transfection, Over Expression, Expressing, Knockdown, Quantitative RT-PCR, Comparison

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 4. QKI-6 acts upstream of HDAC7 and directs its splicing. (A) Conventional PCR was carried out from cDNA of cell lysates overexpressing QKI-6 with primers designed to detect levels of HDAC7 splicing (HDAC7s; 314 base pairs) versus unspliced HDAC7 (HDAC7u; 371 base pairs). Overexpression of QKI-6 leads to increases in both spliced and unspliced HDAC7. (B) Effect of QKI-6 overexpression on the level of spliced HDAC7 mRNA was quantified by qRT-PCR. (C,D) Knockdown of QKI on day 3 of SMC differentiation for 72 h led to a decrease in levels of spliced HDAC7 (C); this is quantified by qRT-PCR (D). (E) To verify the QKI-6- and HDAC7-mediated mechanism of SMC differentiation and to confirm that QKI-6 lies upstream of HDAC7, HDAC7 was knocked down in day 5 miPS-SMCs, followed by QKI-6 overexpression on day 6 alongside relevant combinations of Ex-mCherry and NT controls. Cells were harvested after 48 h and qRT-PCR was carried out. Upon knockdown of HDAC7, QKI-6 overexpression was no longer able to exert its effect on inducing HDAC7, HDAC7 splicing or calponin expression. (F) Overexpression of QKI-5 in day 3 differentiating miPS-SMCs led to a significant reduction in HDAC7 splicing, but was unable to induce SMC marker expression as previously shown by QKI-6 overexpression. Differentiation was also not shown to be directed to an EC lineage upon overexpression of QKI-5 since endothelial cell markers were not observed. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Over Expression, Quantitative RT-PCR, Knockdown, Expressing, Marker

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 5. QKI-6 binds directly to HDAC7 intron 1. (A) RBPmap software revealed a conserved QKI- binding motif that was predicted to align to intron 1 of HDAC7. (B) Specific primers were designed to incorporate this binding motif and amplify a region of 249 base pairs. (C) RNA immunoprecipitation was carried out on miPS- SMCs transduced to overexpress QKI-6 on day 4 of differentiation for 48 h. Pulldown by QKI-6 antibody showed a direct binding to HDAC7 intron 1 through conventional PCR of the resultant cDNA. Image shown is representative of three independent experiments. (D) Relative band intensity of IgG versus QKI-6 pulldown was quantified using ImageJ. Data are mean±s.e.m. (n=3). ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Software, Binding Assay, RNA Immunoprecipitation

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 7. Molecular mechanisms of QKI-directed vascular cell differentiation. Lineage-specific growth factors dictate the expression of the QKI alternative splicing isoforms QKI-5 and QKI-6. VEGF induces the transcription factor ETS-1, promoting QKI-5 binding to STAT3, STAT3 stabilisation and phosphorylation, VEGFR2 activation and VE-cadherin stabilisation. PDGF-BB induces QKI-6 binding to intron 1 of HDAC7, leading to its splicing and promotion of the SRF– myocardin SMC differentiation cascade. Combining QKI-derived vascular cells could be used in numerous applications such as tissue engineering, drug testing and disease modelling.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Cell Differentiation, Expressing, Alternative Splicing, Binding Assay, Phospho-proteomics, Activation Assay, Derivative Assay