Journal: bioRxiv
Article Title: Senescent cell survival relies on upregulation of lysosomal quality control mechanisms
doi: 10.1101/2025.03.31.646397
Figure Lengend Snippet: (A) Proliferative and senescent cells were fixed and immunostained for antibodies against PI4K2A (green) and LAMP1 (magenta), scale bar 10 μm. Colocalisation between PI4K2A and LAMP1 (B) was analysed by Pearson’s correlation. Error bars represent SEM; n = 5 independent experiments with at least 6 fields of view per experimental repeat. Student’s t test (unpaired). (C) Proliferative and senescent cells were incubated with 50 μg/ml cycloheximide (CHX) for the timepoints indicated. The abundance of PI4K2A and the indicated proteins were assessed by western blot. (D) mRNA levels of senescent cells, normalised to PUM1 and presented relative to proliferating cells (dashed line) n = 3 independent experimental repeats for PI4K2A and ULK3 and n = 2 independent experimental repeats for CTSB and TFE3 . (E) Representative immunofluorescence image of proliferative and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2 immunostained for an antibody against LAMP2 (magenta), scale bar 10 μm. (F) Colocalisation between GFP-P4M-SidMx2 and LAMP2 was analysed by Pearson’s correlation. Error bars represent SEM; n = 3 independent experiments with at least 32 cells per experimental repeat. Student’s t test (unpaired). (G) Representative movie stills of live-cell imaging experiment showing LysoTracker-labelled lysosomes (magenta) in proliferating and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2. Scale bar, 10 μm (H) The speed of PI(4)-positive and -negative LysoTracker-labelled lysosomes (μm/frame) are displayed. The mean ± SEM speed from 6 movies per condition are compared by two-way ANOVA followed by Šídák’s multiple comparison. (I) PI(4)P-positive lysosomes in senescent cells display higher MagicRed Cathepsin B activity compared to PI(4)P-positive lysosomes in proliferating cells. Representative movie stills of live-cell imaging experiment showing MagicRed Cathepsin B-labelled lysosomes (red) in proliferating and senescent cells expressing the PI(4)P sensor GFP-P4M-SidMx2. Scale bar, 10 μm. (J) Quantification of PI(4)P-positive and -negative MagicRed Cathepsin B activity in proliferating or senescent cells. Quantification represents mean ± SEM; n = 2 with ≥ 5 movies analysed per condition; two-way ANOVA followed by Šídák’s multiple comparison. *, P < 0.05; **, P < 0.01; ***, P < 0.001;
Article Snippet: The following primary antibodies were used: mouse monoclonal antibodies raised against Lamp1 (H4A3, DSHB, 1:2000), PI4K2A (sc-390026, Santa Cruz, 1:2000), β-Actin (8H10D10, 3700, Cell Signalling Technology, 1:5000), GAPDH (D4C6R, 97166, Cell Signalling Technology, 1:2000), integrin beta 1/CD29 (610467, BD Biosciences, 1:2000), transferrin receptor/CD71 (3B8 2A1, sc-32272, Santa Cruz, 1:1000), GFP (7.1/13.1, Roche, 11814460001, 1:2000), rabbit monoclonal antibodies raised against cathepsin B (D1C7Y, 31718, Cell Signalling Technology, 1:2000), CI-MPR (EPR6599, 124767, Abcam, 1:2000), Lamp1 (D2D11 9091, Cell Signalling Technology, 1:2000), phospho-S6 Ser235/236 (4856, Cell Signaling Technology, 1:2000), S6 (2217, Cell Signaling Technology, 1:1000), phospho-ULK1 Ser757 (D7O6U, 14202, Cell Signaling Technology, 1:1000), ULK1 (D8H5, 8054, Cell Signaling Technology, 1:1000), integrin alpha 5 (EPR7854, ab150361, Abcam, 1:2000), rabbit polyclonal antibodies raised against PI4K2A (15318-1-AP, Proteintech, 1:1000), LDLR (10785-1-AP, Proteintech, 1:2000), CD36 (18836-1-AP, Proteintech, 1:1000), VAPA (15275-1-AP, Proteintech, 1:2000).
Techniques: Incubation, Western Blot, Immunofluorescence, Expressing, Live Cell Imaging, Comparison, Activity Assay
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