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. 2021 Mar 5;24:337–351. doi: 10.1016/j.omtn.2021.03.002

Figure 5.

Figure 5

AR coregulatory gene downregulation and decrease in PCa cancer cell viability are shRNA seed mediated

(A) Relative percent viability and TMEFF2 mRNA expression in LNCaP cells transfected with siTMEFF2-4 and control siRNAs (siNon-target; negative seed controls: siTMEFF2-4+3, siTMEFF2-4+5, 5p-siTMEFF2-4; positive seed control: si634). 5p designates an ON-TARGET-Plus modification (Dharmacon) that reduces off-target effects by reducing miRNA-like gene downregulation. Cell viability was determined by trypan blue. mRNA expression was determined by qRT-PCR. Viability measurements, cell pictures, and RNA extractions were done 72 h after siRNA transfections N = 4, error bars ± SD, ∗p < 0.05 compared to siNon-target. Bars with an asterisk (∗) designate significant differences (p < 0.05) relative to siTMEFF2-4. Significance was determined by t test. (B) Western blot analysis showing caspase-3 cleavage and TMEFF2 protein expression in lysates from LNCaP cells transfected with the designated siRNAs. Lysates were obtained 72 h after siRNA transfections. Calnexin was used as loading control. (C) Relative androgen responsive, AR coregulator, and AR mRNA expression in LNCaP cells transfected with siTMEFF2-4 and control siRNAs. RNA was extracted 72 h after siRNA transfections. mRNA expression was determined by qRT-PCR. n= 4, error bars ± SD, ∗p < 0.05 determined by t test.