Figure 1: LINE-1 and Alu RNA are enclosed within autophagosomes. | Nature Communications

Figure 1: LINE-1 and Alu RNA are enclosed within autophagosomes.

From: Autophagy supports genomic stability by degrading retrotransposon RNA

Figure 1

(a,b) Two representative images of fluorescent microscopy of LC3 and LINE-1 fused to six MS2-binding sites detected with MS2-GFP. (c) Fluorescent microscopy of LC3 and Alu fused to six MS2-binding sites detected with MS2-GFP. Li’s correlation coefficient of co-localization (0.0232, s.e.m. 0.048). (d) Relative number of foci of LINE-1-MS2, or number of foci of LINE-1-MS2 co-localized with LC3 per cell in randomly selected fields of cells treated with BAF1 (20 h, 400 nM). n=25 cells, total number of LINE-1-MS2 foci: control=104, BAF=175, total number of LINE-1-MS2 foci co-localized with LC3: control =68, BAF 167. *Total P=0.015, Co-localized with LC3 P=5 × 10−8, t-test). Li’s correlation coefficient of co-localization (without BAF 0.19 s.e.m. 0.027; with BAF 0.213, s.e.m. 0.02). (e,f) Two representative images of fluorescent microscopy of FISH probes recognizing endogenous LINE-1 RNA and LC3. (g) Three-dimensional reconstruction of confocal microscopy z-stacks of endogenous LINE-1 RNA and LC3 prepared in Imaris. (h) Western blot of fractions enriched in autophagosomes compared with total cell extracts and ER/mitochondria-enriched fractions. RT-qPCR analysis of mRNA levels of two transmembrane proteins (GLUT4 and APP) in total cell RNA and autophagosome-enriched fractions. n=3, P>0.05. (i) RT-qPCR analysis of LINE-1 ORF1 and AluYa5 in RNAse-treated autophagosome-enriched fractions or total cell RNA. n=3, *P=0.043 LINE-1, P-0.01 AluYa5, t-test (j) Model of system to measure degradation of GFP-tagged substrates by autophagy. (k) Western blot analysis of GFP and alpha-tubulin (control) in cells transfected with MS2-GFP or LINE-1-MS2 and MS2-GFP and treated with control or Bafilomycin (2 h, 400 nM). All error bars represent s.e. of the mean. All experiments were replicated a minimum of three times.

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