Fig 1.
Construction of vectors and generated cell line.
(A) Vector map of AAVS1-TRE-TurboID-rtTA, zoom panel shows restriction sites presents in N-terminal of TurboID-HA. Vectors with U2AF2 and GFP showed on right side. (B) Immunolabeling of hiPSC iU2AF2-TurboID and hiPSC iGFP-TurboID stained with anti-HA, comparing induced and non-induced cells. Induction was performed with 1 µg/mL of doxycycline for 24 hours. Merge performed with DAPI stain for nucleus labeling. Scale: 50 µm C. Confocal microscopy for both cell lines stained with anti-HA after 24 hours of induction. White arrow pointing the HA-tagged fusion proteins. Merge performed with DAPI stain for nucleus labeling. Scale: 10 µm.
Fig 2.
Inducible expression and biotinylation activity of TurboID.
(A) Western blot showing time-course induction, as indicated above, stained with anti-HA and normalized with anti-Actin. The top panel shows hiPSC iU2AF2-TurboID, and the bottom panel shows hiPSC iGFP-TurboID. (B) Western blot of iGFP and iU2AF2 samples after 4 hours of induction. NI – non-induced; IND – induced; + Bio – induced and supplemented with 500 µM biotin. Biotinylated proteins were stained with streptavidin-AlexaFluor-647, and images were acquired using the iBright Imaging System.
Fig 3.
Mesoderm differentiation of hiPSC iU2AF2-TurboID and hiPSC iGFP-TurboID.
(A) Experimental design for PPI in mesoderm differentiation. (B) Mesoderm markers by qPCR comparing pluripotent state and mesoderm for both iU2AF2 and iGFP-TurboID cell lines. Statistical analysis with t-student’s test. ***p < 0.001. (C) Immunolabeling of differentiated mesoderm from hiPSC iU2AF2-TurboID and hiPSC iGFP-TurboID stained with anti-HA after 4 hours of induction with 1 µg/mL of doxycycline. Merge performed with DAPI stain for nucleus labeling. Scale: 100 µm. Right panel show a zoom region.
Fig 4.
Proteomic analysis for U2AF2-TurboID and GFP-TurboID proximity labeling proteins.
(A) Protein identified by comparing U2AF2 and GFP samples in pluripotent (left panel) and mesoderm (right panel) Axis Y threshold: p-value≤0.05 (-log p-value≥1.3); Axis X threshold: difference of Log2ΔLFQ≥2. Gene names are next to their representative dots. (B) Protein identified by comparing U2AF2 and GFP samples in pluripotent (left panel) and mesoderm (right panel). Axis Y threshold: pvalue≤0.05 (-log p-value≥1.3); Axis X threshold: difference of Log2ΔLFQ≥1. Gene names are next to their representative dots. (C) Protein identified by comparing of U2AF2 in pluripotent and mesoderm. Negative difference means more peptides in pluripotent, positive difference means more peptides in mesoderm. Axis Y threshold: p-value≤0.05 (-log p-value≥1.3); Axis X threshold: difference of Log2ΔLFQ≥2. Gene names are next to their representative dots.
Table 1.
List of U2AF2-interacting proteins identified by proximity labeling comparing the pluripotent and mesoderm stages.
Fig 5.
Interactome of U2AF2 in pluripotent and mesoderm stages.
Interaction network created with STRING webtool and Cytoscape software. Panel in dashed line showing the directly links proteins with U2AF2 in pluripotent (A) and mesoderm (B) stages. Black lines represent direct interactions with U2AF2, while gray lines indicate interactions among other identified proteins. Gene Ontology table generated with Enrichr webtool using the genes present in whole interaction network.