Fig. 4. Aberrant TE mobilization leads to the cytoplasmatic accumulation of RNA–DNA hybrids in FAD hippocampal progenitors.

a Immunofluorescence for RNA–DNA hybrids (S9.6 antibody, pink signal) displays an accumulation of RNA–DNA hybrids in the cytoplasm of FAD hpNPCs. Scale bar 50 µm, 40× magnification. DAPI staining on nuclei in blue. Each IF was replicated three times with similar results (n = 3 independent experiments). b Quantification of immunostaining in panel (a). The IF data are quantified using a Superplot, which concisely visualizes individual data points and their averages. The distinct combinations of colors and shapes indicate the three independent experiments performed. Each small dot in the graph corresponds to a specific data point representing an analyzed image or cells. The larger dots represent the average values calculated from the respective data points. A Repeated measures ANOVA test was used for the comparison between the two groups. A value of P < 0.05 was considered significant; **P < 0.01 (RNA;DNA hybrids P = 0.00329). Error bars report the standard error. c Violin plot of log₂(TPM) for RNaseH in CTRL and FAD hpNPCs. d qPCR analysis on the cytoplasmic fraction for a group of TEs selected among those previously identified as aberrantly active in FAD hpNPC. Primers for HERVK/LTR5HS target individual ORFs from the LTR; Primers for L1-ORF1 target a conserved region in ORF1 of 6x; the other L1 primers target the L1PA2 family; the LH2/LH3 primers target the end of the 3’ UTR; The LH1/LH2 primers target the 5’ of the other amplicon. Each dot in the bar plot represents an experiment (n = 7 independent experiments). A two-sided t-test was used for the comparison between the two groups. A value of P < 0.05 was considered significant; *P < 0.05 (HERVK ENV P = 0.013637; HERVK POL P = 0.012468); n.s., not significant (LTR5HS P = 0.245766; LH3/LH2 P = 0.147473; LH2/LH1 P = 1; L1 ORF1 P = 0.366467; HERVK GAG P = 0.184794). Error bars report the standard error.