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. 2022 Apr 7;50(9):4917–4937. doi: 10.1093/nar/gkac233

Figure 3.

Figure 3.

Effects of YY1 cIDR and H-cluster mutagenesis on its biological functions. (A) Ectopic YY1 expression with simultaneous endogenous YY1 knockdown. MDA-MB-231 cells with DOX-induced sh-YY1 targeting the 3′-UTR of the YY1 mRNA were infected by lentivirus expressing 3 × Flag-YY1 WT and mutants, or a vector. Cell lysates were analyzed by Western blot using indicated antibodies. (B) Effects of YY1 mutations on cell viability. MDA-MB-231 and MCF-7 cells expressing 3 × Flag-YY1 WT and mutants with endogenous YY1 knockdown were evaluated by WST-1 assays to determine cell viability. **P < 0.01; ***P < 0.001. (C and D) Mouse xenograft tumor formation to evaluate the function of YY1(H-A) mutant. MDA-MB-231 cells (4 × 106) carrying DOX-inducible sh-YY1-3′-UTR and infected by lentivirus carrying an empty vector, or expressing WT YY1 or YY1(H-A). In (C), the growth curves of mouse xenograft tumors were generated by measuring tumor sizes twice a week and then calculating tumor volumes. In (D), IF staining and RT-qPCR analyses of frozen xenograft tumor samples. The IF images of YY1, Ki-67 and FOXM1 antibodies and their merged images with DAPI are presented, with the quantitation shown at the upper right panel. The RT-qPCR data of YY1 and FOXM1 mRNA expression normalized to GAPDH levels at the lower right panel. (E–H) IDR fusion studies to evaluate the function of the H-cluster of YY1. In (E), the diagrams of fusion proteins by conjugating the FUS IDR (FUSIDR) and TIA1 IDR (TIA1IDR) to the N-terminus of YY1(H-A) mutant are displayed. In (F), representative fluorescence and DIC images of EGFP-conjugated proteins as labeled on top are presented. Quantification of droplet's numbers and area is shown at the low panel. In (G), live U2OS cells transfected with EGFP-conjugated proteins as labeled at left are shown. In the quantifications of (F) and (G), P values for the comparison of different groups are labeled. In (H), cell viability was determined by WST-1 assays for the MDA-MB-231 cells with endogenous YY1 knocked down by sh-YY1-3′-UTR and infected by lentivirus carrying an empty vector or expressing Flag-YY1 WT, H-A mutant, or its fusion proteins. **P < 0.01; ***P < 0.001; n.s.: not significant.