Fig 1: Characterization of 27nt-RNAs and their association with macronuclear sequences. a Time course of small RNA fragment size distribution (10–50nt) and associated stages of macronuclear development showing the dominance of 27nt—RNAs (light blue columns). Red columns mark 21–22nt-RNAs. The y-axis shows the normalized read quantity (× 105). b The illustration shows the coverage of 27nt-RNAs (light blue bars), 21–22nt-RNAs (red bars) and mRNA reads (green curves) on four exemplary nanochromosomes with complete telomere-to-telomere sequence (red contigs). Notably, the mapped area frequently includes introns (1.95% of 27nt-RNA/1.43% of 21–22nt-RNA). The position of predicted full-length transcripts (yellow arrows) and coding sequences (CDS; green arrows, occasionally interrupted by introns [red arrows]) is illustrated. The bars below (purple) show furthermore the coverage of 27nt-RNAs, which were purified from pulled-down RNA/DNA hybrids using the mouse anti-RNA/DNA hybrid [S9.6] monoclonal antibody (mAb). c Average sncRNA (27nt-RNAs and 21–22nt-RNAs) read counts matching per contig. d Combined correlation analyses between mRNA reads (transcript), 27nt-RNAs and the relative nanochromosome copy numbers. e PAR-CLIP revealed a prominent RNA band well in agreement with the expected size of a PIWI1/27nt-RNA complex (sncRNA1)), whereas a smaller, at best very faint band could correspond to the expected size of a PIWI1/21–22nt-RNA complex (sncRNA2). f The read coverage is shown on the left for 4 exemplary nanochromosomes for 27nt-RNAs enriched in immunocomplexes using the mouse anti-RNA/DNA hybrid [S9.6] mAb (Kerafast #ENH001) (purple signals) or, respectively, rabbit anti-PIWIL1 polyclonal antibody (pAb) (Abcam #ab12337) (light blue signals). Symbolism and colour coding of the nanochromosome annotation equals (b). The chart (right) shows the results of correlation analyses between the reads obtained from 27nt-RNA-seq (x-axis: RNA/DNA-IP; y-axis: PIWI1-IP)
Fig 2: Betulinic acid effects on Piwil1 gene expression. Piwil1 mRNA expression was assessed in the presence and absence of serum and BA in the lung cancer cell lines A549 (A) and NCI-H1299 (B). RT-PCR data show that piwil1 expression was down-regulated by BA in the absence and presence of serum. PCR reactions were set in triplicates and Cyclophillin was used as an internal loading control and used to normalize the relative expression levels using the 2−ΔΔ method (26). RT-PCR reaction were run at three independent times.
Fig 3: PIWIL1 promotes the growth of xenograft tumors and enhances resistance of MM cells to chemotherapy in vivo. (A) Tumor volume of NOD/SCID mice xenografted with different groups of transfected RPMI-8226 cells. (B) Tumor growth curves of NOD/SCID mice after transfected RPMI-8226 cell injection. (C, D) Tumor volume and tumor growth curves of xenograft tumors after bortezomib treatment. (E) Representative immunohistochemical staining of p-mTOR, P62, optineurin, Nanog, OCT4, and Ki67 in tumor xenografts. Data are from representative images of three separate experiments. (**P < 0.01, NS, p > 0.05).
Fig 4: The feline genome encodes multiple transcript variants of PIWIL1. Diagram of encoded transcript variants from the feline genome denoting included and excluded exons in each case. Resulting protein isoforms, PIWIL1-isoform 1 and PIWIL1-isoform 2, respectively, are labeled to indicate important protein domains.
Fig 5: (A). PIWI protein expression in ovarian tissue. (B). PIWI protein expression statistics
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