Piwi4 and Piwi5 adhere to different targeting rules. (A) Northern blot analysis comparing the levels of the two propiR1 isoforms in Piwi4 and Piwi5 single and double knockdown (dsPiwi4, dsPiwi5) to a control knockdown (dsRNA targeting Luciferase, dsLuc). U6 snRNA serves as a loading control. The numbers below the blot are the quantified signal for each of the individual propiR1 isoforms, normalized to the U6 loading control, with the dsLuc control set to 1. (B) Luciferase assay of reporters bearing a fully complementary propiR1 target (full), or the endogenous target site of lnc027353 after single or double knockdown of Piwi4 and Piwi5. A propiR1 target site in which residues t4–6 were mutated (mut4–6) serves as control. Luciferase activity was normalized to the mut4–6 control treated with the same dsRNA. Shown is a representative of four independent experiments, each performed with three biological replicates (the other experiments are shown in Supplemental Fig. S6). Bars represent mean ± SD. Asterisks indicate statistically significant differences of the indicated reporters upon PIWI-gene knockdown compared to dsGFP-treatment (unpaired two-tailed t-tests with Holm–Sidak correction; [*] P < 0.05, [**] P < 0.005, [***] P < 0.0005). (C) Relative expression of the propiR1 target gene lnc027353 upon Piwi4 and Piwi5 single and double knockdown in Aag2 cells, measured by RT-qPCR. Luc knockdown serves as a control and was used in single knockdowns to equalize the total amount of dsRNA per condition. Data represent the mean ± SD of three biological replicates. Asterisks indicate statistically significant differences in lnc027353 expression compared to dsLuc (unpaired two-tailed t-tests with Holm–Sidak correction; [***] P < 0.0005).
