Fig. 2: ANC-1 and nesprin-2 regulate autophagy.
From: Nucleophagy delays aging and preserves germline immortality
a, Epifluorescence microscopy head region imaging of day 2 WT worms and atg-2(bp576) and daf-2(1370) mutants, expressing the indicated reporter genes, subjected to RNAi-mediated silencing of the anc-1 gene. The arrows indicate punctate structures. Scale bar, 20 μm. b, Quantification of GFP puncta per head and puncta area from a. The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. ****P < 0.0001 using an unpaired t-test; n ≥ 17 worm head regions. c, Epifluorescence microscopy imaging of day 1 punc-54Q40::YFP worms on RNAi-mediated silencing of the indicated genes. Scale bar, 20 μm. d, Quantification of Q40::YFP puncta per worm from c. The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. ****P < 0.0001 using a one-way ANOVA; n ≥ 29 worms. e, Confocal microscopy immunofluorescence imaging of LC3B in control and BafA1-treated, WT and nesprin-2−/− E14.5 MEFs. Scale bar, 20 μm. f, Quantification of LC3B puncta number per cell from e. The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. NS ≥ 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001 using a two-way ANOVA; n ≥ 22 cells. g, Western blot analysis of endogenous LC3B in control and Earle’s balanced salt solution-starved WT and nesprin-2−/− E14.5 MEFs, with or without BafA1 treatment. h, Quantification of the LC3B-II/I ratio. Mean ± s.d. of four biological replicates. i, Confocal microscopy imaging of control and BafA1-treated, WT and nesprin-2−/− E14.5 MEFs stained with DAPI (blue) and immunostained for p62 (red). The dashed lines represent nuclei. Scale bar, 20 μm. j, Quantification of nuclear p62 fluorescence intensity in control and BafA1-treated, E14.5 MEFs from i. The box plot represents the 25th–75th percentiles, the line depicts the median and the whiskers show the min–max values. NS ≥ 0.05, ****P < 0.0001 using a two-way ANOVA; n = 19 cell nuclei.
