1. Fig. 5. Wisper is associated with TIA1-related protein and regulates lysyl hydroxylase 2 expression.
Wisper is associated with TIA1-related protein and regulates lysyl hydroxylase 2 expression. (A) Proteins enriched after pulldown using biotinylated Wisper and identified by mass spectrometry. The x axis shows the protein enrichment in the Wisper group as compared to the control antisense Wisper group; the y axis shows the −log P value determined by Fisher’s test. (B) Amino acid composition of the protein TIAR. The detected amino acids by mass spectrometry are highlighted in yellow (103 of 392 amino acids; 26% coverage). (C) Protein quantification of TIAR by Western blotting in the pulled-down protein fraction. Purified TIAR is used as positive control. Graph shows means ± SEM (n = 3). P values were determined by Student’s t test. A.U., arbitrary units. (D) Quantification of RNA after immunoprecipitation using an immunoglobulin G (IgG) directed against TIAR or a control IgG. Protein lysate was produced from CFs after transfection with GapmeRs targeting Wisper (GM-Wisper) or scrambled GapmeRs (GM-Scr) (10 nM, 48 hours). Graph shows means ± SEM (n = 6). P values were determined by two-way ANOVA (Fisher’s test). Ab, antibody. (E) Plod2 expression in adult CFs after GM-Wisper, GM-Wisp2, or GM-Scr transfection (10 nM, 48 hours). Bars represent means ± SEM (n = 3). P values were determined by one-way ANOVA. (F) Immunohistochemistry analysis of TIAR expression in CFs transfected with GapmeRs targeting Wisper (GM-Wisper) or left untreated (none; 10 nM, 48 hours). TIAR, green; actin, red; 4′,6-diamidino-2-phenylindole (DAPI), blue. (G) Confocal microscopy analysis of TIAR subcellular localization in CFs treated as in (F). (H) Percentage of CFs with nuclear TIAR staining after GM-Wisper treatment at various doses. Bars represent means ± SEM (n > 6). P values were calculated by two-way ANOVA (Fisher’s test).
Rudi Micheletti et al., Sci Transl Med 2017;9:eaai9118
Published by AAAS