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. 2024 Apr 23;35(2):102193. doi: 10.1016/j.omtn.2024.102193

Figure 2.

Figure 2

DUPA-miR-34a uptake and internalization by prostate cancer cells is DUPA and PSMA mediated

(A) Representative confocal images of LNCaP cells treated with 100 nM DUPA-miR-34a-Atto.647N or miR-34a-Atto.647N (no DUPA) for 4 h, Scale bar, 10 μM. (B) Representative confocal images of LNCaP cells treated with 100 nM DUPA-miR-34a-Atto.647N (red) at different time points after treatment. Scale bar, 10 μM. (C) Quantification of miR-34a uptake in PSMA positive LNCaP cells using qRT-PCR. LNCaP cells were treated (100 nM) or transfected (+Lipo, 10 nM) with DUPA-miR-34a or DUPA-NC (siLuc2, NC) for 18 h (top). PSMA negative A549 cells treated with DUPA-NC or DUPA-miR-34a, or transfected with a NC miRNA (miR-NC) or with a commercial miR-34a mimic. One representative experiment is depicted from a total of three biological experiments. ∗∗p < 0.01, ∗∗∗∗p < 0.0001, one-way ANOVA. Error bars are mean ± SD. (D) Targeted silencing of miR-34a Renilla sensor after transfection of LNCaP-miR-34a sensor cells with 50 nM DUPA-miR-34a, miR-34a duplex, or respective controls for 24 h. One representative experiment is depicted from a total of three biological experiments. ∗∗∗∗p < 0.0001, one-way ANOVA. Error bars are mean ± SD. (E) Dose-dependent response of LNCaP-miR-34a sensor cells following treatment with DUPA-miR-34a, miR-34a duplex, or respective controls for 96 h (n = 2). ∗∗p < 0.01, one-way ANOVA. Error bars are mean ± SD. (F) DUPA-miR-34a competition assay following treatment of LNCaP-miR-34a sensor cells with DUPA-miR-34a (100 nM, 96 h) in the presence of 10-, 100-, or 500-fold higher concentrations of PMPA (n = 2). ∗∗p < 0.01, one-way ANOVA. Error bars are mean ± SD. (G) Representative confocal images of LNCaP cells treated with 100 nM DUPA-miR-34a-Atto.647 (pink) for the indicated time points. Late endosomes and lysosomes were stained with LysoTracker (green). Nuclei were stained by Hoechst (blue). Insets in the top left corner of DUPA-miR-34a-treated samples represent scatterplot of LysoTracker vs. miR-34a fluorescence intensities. Appearance of intracellular white fluorescence indicates late endosomal/lysosomal localization of DUPA-conjugates. Scale bar, 10 μM. (H) Representative graph showing quantification of miR-34a colocalization with late endosome/lysosomes at the indicated times by Pearson’s correlation coefficient analysis (n = 2). ∗p < 0.00001, two-tailed unpaired t test. Error bars are mean ± SD.