Skip to main content
. 2018 Dec 18;129(2):583–597. doi: 10.1172/JCI124011

Figure 7. The CLK inhibitor TG003 suppresses IVS4+866 C>T–induced PE inclusion to restore the production of a functional NEMO protein.

Figure 7

(A) Genomic sequence surrounding the 44-bp PE of IKBKG. Black bars indicate the RNA sequences used for the pulldown assays (PE-5′ and PE-3′, WT, and IVS4+866 C>T). (B) Western blot of RNA-pulldown products (WT and IVS4+866 C>T) for U1snRNP subunits (U1-70k, U1-A, U1-C, and SmB/B′). (C) Western blot of RNA-pulldown products (PE-5′, PE-3′, WT, and IVS4+866 C>T) for phosphorylated SR proteins. (D) Western blot of P3-derived iPSC-Mϕ transfected with nonspecific siRNA or SRSF1-specific or SRSF6-specific siRNA. β-Actin served as the internal control. (E) TNF-α production by P3-derived iPSC-Mϕ transfected with nonspecific, SRSF1-specific, or SRSF6-specific siRNA for 72 hours was evaluated 4 hours after stimulation with LPS and IFN-γ (n = 3). (F) Diagram of the SPREADD reporter for IKBKG exons 4–5 with the IVS4+866 C>T mutation. The GFP signal indicates IKBKG exon 4^5 splicing, whereas the RFP signal indicates inclusion of the 44-nt PE. (G) The intensities of green and red fluorescence were quantified in HeLa cells transfected with the IKBKG (IVS4+866 C>T) SPREADD vector and treated with small-molecule compounds (TG003, TG009, and SRPIN340) or not (0.1% DMSO) for 24 hours. Dot plots represent the GFP/RFP ratio of 6 random fields (80–100 fluorescence-positive cells/field) from a single experiment. **P < 0.001. (H) Representative fluorescence micrographs of cells treated with 10 μM TG003 or 0.1% DMSO following transfection with the IKBKG (IVS4+866 C>T) SPREADD vector. Nuclei were counterstained with Hoechst 33342. Scale bars: 200 μm. (I) Western blot of NEMO and β-actin for protein extracts from P3-derived or control iPSC-Mϕ. (J) TNF-α production by P3-derived iPSC-Mϕ stimulated with LPS and IFN-γ for 4 hours, with or without TG003 pretreatment. Data are presented as the mean ± SD of 3 independent experiments using a representative clone. *P < 0.05, by 1-way ANOVA followed by Dunnett’s test.