Fig 1: PIWI proteins were dysregulated in postmortem sALS samples. A RT-qPCR for PIWIL1, PIWIL2, PIWIL3, PIWIL4 expression of mRNA using the ΔCT method. RT-qPCR using the △CT method for PIWIL1 and PIWIL4 showed significant differences between ALS and control samples, while PIWIL2 and PIWIL3 showed non-significant differences. B RT-qPCR results of postmortem PIWIL1 and PIWIL4 expression using a control pool for the standard curve method. One-tailed t-test and the Wilcoxon test were used for statistical calculations. PIWIL1 was upregulated 1.2–1.9-fold in sALS patients compared to the mean of control samples, by the standard curve method. C Western blot analysis of PIWIL1 and PIWIL4 in postmortem samples. The increase of PIWIL1 protein and the decrease of PIWIL4 protein were confirmed in ALS samples. D Quantitative analysis of PIWIL1 and PIWIL4 in a Western blot normalized to GAPDH. The increase of PIWIL1 protein and the decrease of PIWIL4 protein were statistically confirmed. ns: not significant. *p < 0.05, **p < 0.01
Fig 2: Rt‒PCR of the relative expression of PIWIL1, PIWIL2, and PIWIL4 in different ovarian tissues.
Fig 3: In CTCL, GTSF1 does not regulate retrotransposons. (A) Western blot analysis of piRNA pathway elements TDRD9, PIWIL4, DDX4 and PIWIL2 (top) and of retrotransposon L1 ORF1p (bottom) in CTCL cell lines. Protein from mouse testis is used as a positive control (top), protein from cell line Calu‐6 is used as a positive control (bottom) and protein from cell line N/Tert‐1 (top and bottom) is used as a non‐malignant control. GAPDH was used as a loading control and is presented for each membrane probed. (B) Western blot analysis of GTSF1 after lentiviral shRNA‐mediated knockdown in the three CTCL cell lines Mac2A, MyLa and SZ4. GAPDH was used as a loading control. (C) Western blot analysis of L1 ORF1p after GTSF1 knockdown in CTCL cell lines. Protein from cell line Calu‐6 is used as a positive control. GAPDH was used as a loading control. (D) Luciferase activity from retrotransposition assay in Relative Luminescence Units (RLU). Each assay was performed with two reporter plasmids, pYX014 and pYX017, for each cell line. Firefly luminescence was corrected with the corresponding Renilla luminescence value and the retrotransposition incompetent plasmid pYX015 is used to normalise luminescence values. Differences between SCR (dark blue bars) and shGTSF1 (light blue bars) were evaluated with unpaired two‐tailed t test. Data are presented as means of three replicates ± SD.
Supplier Page from Abcam for Anti-PIWIL2 antibody