Figure 2: Inhibiting autophagy increases levels of LINE-1 and Alu RNA and their corresponding genomic insertions. | Nature Communications

Figure 2: Inhibiting autophagy increases levels of LINE-1 and Alu RNA and their corresponding genomic insertions.

From: Autophagy supports genomic stability by degrading retrotransposon RNA

Figure 2

(a,b) RT-qPCR analysis of levels of (a) LINE-1 RNA (5′UTR, ORF1, n=3, *P=0.009 LINE-1 5′UTR, P=0.03 LINE-1 ORF1, t-test) and (b) AluYa5 and AluYb8 in cells (n=3, *P=0.0018 AluYa5, P=2 × 10−6 AluYb8, analysis of variance (ANOVA)) treated with siRNA-targeting ATG5 or control (10 nM, 48 h) (c) RT-qPCR analysis of AluYa5 and AluYb8 in 250–350 nt RNA isolated from cells treated with siRNA-targeting ATG5 or control (10 nM, 48 h, n=3, *P=0.046 AluYa5, P=0.043 AluYb8, ANOVA). (d) RT-qPCR analysis in cells treated with dimethylsulphoxide or Bafilomycin (400 nM) and actinomycin D. (5 nM, n=3, *P=0.01 LINE-1 both 4 and 8 h, P=0.04 AluYb8, ANOVA). (e) RT-qPCR analysis of pulse-labelled RNA recovered 0 and 4 h after pulse in cells transfected with siRNA-targeting ATG5 or control (10 nM, n=3, *P=0.01 both LINE-1 and AluYb8). (f) Percent of cells expressing GFP when co-transfected with LINE-1-RP GFP reporter of retrotransposition and siRNA-targeting ATG5 or control. Results are normalized to similar experiments with a retrotransposition-incompetent control (n=3, *P=0.045 ATG5-1, P=0.048 ATG5-2, ANOVA). (g) Relative number of cells at experiment’s termination if cells were transfected with siRNA-targeting ATG5 or control. (h) Percent of cells expressing GFP when co-transfected with plasmid expressing GFP and siRNA-targeting ATG5 or control (n=3). (i) Representative plates from Alu-retrotransposition assay transfected with control siRNA or siRNA-targeting ATG5. (j) Relative number of G418-resistant colonies in cells transfected with siRNA-targeting ATG5 or control, Alu-retrotransposition reporter and retrotransposition-enhancer LINE-1 ORF2p (n=3, *P=0.01 ATG5-1, P=0.02 ATG5-2, ANOVA) All error bars represent s.e. of the mean. All experiments were performed with a minimum of three biological replicates.

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