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. 2022 Mar 7;12:3687. doi: 10.1038/s41598-022-07642-1

Figure 1.

Figure 1

UCHL5 negatively regulates the Wnt/ß-catenin signaling pathway. (a) TOPflash assay using HeLa cells. Cells were transfected with the indicated plasmids (50 ng TK-Renilla reporter; 250 ng TOPFlash; 250 ng FOPFlash; 1 μg and 2 μg pCS2 + UCHL5). Then, Wn3a conditioned media (Wnt3a CM) was treated for 16 h. (b) Quantitative PCR (qPCR) analysis for Axin2, pCS2 + UCHL5 was introduced into HeLa and MCF7 cells. Then, Wnt3a CM was treated for 16 h. GAPDH was used for normalization. (c) Western blot analysis using HeLa cells. Flag-UCHL5 plasmids (2 μg and 4 μg) were transfected with empty vector. Transfected cells were treated with either L- cell control CM (L CM) or Wnt3a CM for 16 h. (d) qPCR analysis for UCHL5. Total RNAs were extracted from control (Co) HeLa and two different stable knockdown HeLa cells (KD1 and KD2). Samples were subjected to cDNA synthesis and qPCR analysis. GAPDH was used for normalization. (e) TOPflash assay using stable HeLa cells. Co, KD1, and KD2 HeLa cells were transfected with TOPflash (200 ng) and TK-Renilla reporter (50 ng). Then, cells were treated with Wnt3a CM for 16 h. (f) qPCR analysis for Axin2 and c-myc. Co, KD1, and KD2 HeLa cells were incubated with Wnt3a CM for 16 h. (g) Western blot analysis using Co and KD HeLa cells. Co, KD1 and KD2 HeLa cells were incubated with Wnt3a CM for 16 h. (h) TOPflash assay using Co and KD1 HeLa cells. Either pCS2 + or pCS2 + UCHL5 (1 μg) was co-transfected with TOPflash reporter (250 ng) and TK-Renilla reporter (50 ng) into cells. For Wnt stimulation, Wnt3a CM (for 16 h), Dvl plasmids (0.5 μg), and ptß-catenin plasmids (0.2 μg) were introduced. The data from TOPflash (a, e, h) and qPCR analyses (b, d, f) are displayed as means ± SD and show a representative of multiple independent experiments (n = 3 biological independent experiments). * P < 0.05, ** P < 0.005. from two-tailed unpaired t-test (a, b,d, e, f, h).