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. 2021 Aug 10;13(16):4025. doi: 10.3390/cancers13164025

Figure 2.

Figure 2

Inhibition of LNCaP or C4-2 spheroid growth and hTERT expression by SAL treatment. Spheroids from both LNCaP (A,B) and C4-2 cells (C,D) were generated and treated with the indicated ligands 1nM R1881 (SAL), 1pM R1881 (LAL), 10nM DHT (DHT SAL), or the DMSO control for 8 days. The representative pictures of the spheroids from LNCaP (A,B) and C4-2 (B,D) were analyzed every second day. The mean ± SEM values were calculated from three independent experiments, whereas for each biological experiment, six technical replicates were performed. The significance of the data from the treatments versus DMSO was analyzed with two-way repeated measure ANOVA with Dunnett’s multiple comparisons test (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001). Both LNCaP (E,F) and C4-2 (G,H) spheroids were cultured for 8 days with the indicated ligands, and were sliced (4 µM thick) and subsequently immunostained for Ki-67 as a proliferation marker (E,G), and finally were analyzed for SA β-Gal activity as a hallmark for senescent cells (F,H). The scale bar is indicated for 100 µm. Spheroids generated from LNCaP (I) or C4-2 (J) cells were analyzed for hTERT mRNA by qRT-PCR. Values were calculated relative to DMSO, which was set arbitrarily as 1. Error bars indicate SEM. *** p ≤ 0.001.