Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: a , Schematic description of the dualPIM system. b , IEM analyses of dualPIM aggregates induced for 30 min, 2 h or 6 h. Cryosections were labelled individually with anti-GFP (30 min and 2 h time points) or in combination with anti-LAMP2 antibodies (6 h time point). Insets are shown below the overviews. Scale bars, 500 nm (overviews) and 250 nm (insets). c , HeLa cells transiently expressing dualPIM were imaged until 16 h after triggering aggregation with rapa2 treatment. Time-lapse images showing multiple fragmentation (white arrows) and degradation (white arrowhead) events are presented in the panel. See also Supplementary Video and Extended Data Fig. . CM, control medium. d , DualPIM cells were treated ± 40 µM VER for 19 h. e , f , Quantification of the average dualPIM puncta size at 19 h ( e ) and degradation rates ( f ). P = 0.0055 ( e ); P < 0.0001 ( f ). g , DualPIM cells were transfected with the indicated siRNAs (also Supplementary Table ) and imaged for 24 h. Representative images at 24 h are shown. h , i , Quantification of dualPIM puncta average size at 24 h ( P values versus siCtr: P = 0.001 (siCtr + BAF); P = 0.024 (siHSP70/siHSPA1A); P < 0.001 (siHSP110); P < 0.001 (siDNAJA2); P < 0.001 (siDNAJB6)) ( h ) and degradation rates ( P values versus siCtr: P < 0.001 (siCtr + BAF); P = 0.002 (siHSP70/siHSPA1A); P < 0.001 (siHSP110); P = 0.004 (siBAG3); P < 0.001 (siDNAJB6)) ( i ) of the experiment are shown. Images in c were acquired using the Nikon Eclipse microscope and images from d and g using the IncuCyte S3 system. Data are presented as mean ± s.d. Error bars represent the s.d. of three ( e , f , h , i , siHSPB1, siHSPB7, siHSP110, siDNAJA1, siDNAJA2, siDNAJB1 and siDNAJB4 condition), four ( h , i , siHSPBP1, siHSP70/siHSPA1A, siBAG3, siDNAJB2 and siDNAJB6 condition), eight ( h , i , siCtr condition) or ten ( h , i , siCtr + BAF condition) independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001. P values were calculated by a two-tailed t -test with Bonferroni correction ( e , f ) or one-way analysis of variance (ANOVA) following Dunnett’s post hoc test ( h , i ). Scale bars, 20 µm, except in a (5 µm).

Article Snippet: Detection of Lamp2 was performed using an anti-Lamp2 antibody (BD biosciences, 555803) follow by a rabbit anti-mouse (Rockland, 610-4120) and protein A-conjugated gold (GFP: 15 nm; LAMP2: 10 nm).

Techniques: Expressing, Control, Transfection, Microscopy, Two Tailed Test