Fig. 5: Depletion of PML or Ubc9 leads to the accumulation of DRiPs inside SGs.
From: Loss of PML nuclear bodies in familial amyotrophic lateral sclerosis-frontotemporal dementia
A HeLa Kyoto cells stably expressing GFP-PML were treated with OP-puro (25 μg/ml) and MG132 (20 μM) for 3 h. Cells were fixed and stained for SUMO1. Nucleic acid was labeled using DAPI. GFP-PML-NBs were automatically segmented, followed by quantification of the enrichment for SUMO1 inside each GFP-PML-NB. Enrichment < 1 corresponds to GFP-PML-NBs devoid of SUMO1; enrichment > 1 corresponds to GFP-PML-NBs colocalizing with SUMO1. The GFP-PML-NB number analyzed was as follows: 2388 (sample 1); 3636 (sample 2); 3549 (sample 3). B HeLa Kyoto cells were lipofected with a non-targeting siRNA control (siRNA control) or with siRNA secific for PML and Ubc9; 72 h post-transfection, cells were treated with OP-puro (25 μg/ml) and MG132 (20 μM) for 3 h. Cells were fixed and subjected to click chemistry to visualize DRiPs, followed by immunostaining with antibodies specific for the SG marker TIA1 and SUMO1. Considering that SUMO1-PML assembles into nuclear bodies, SUMO1 simultaneously enables to visualize SUMO1-conjugated proteins and SUMOylated PML-NBs. Note the three dotted-like structures positive for SUMO1 and DRiPs in the nucleus of cells transfected with siRNA CTL (white dot in the 3x zoom panel). Note also that SGs in these cells do not colocalize with DRiPs (* in the 3x zoom panel). Instead, in cells treated with siRNA PML and siRNA Ubc9, note the absence of SUMO1 and DRiPs positive puncta in the nucleus (* in the 3x zoom panel) and the colocalization of SGs with DRiPs (* in the 3x zoom panel). Nucleic acid was labeled using DAPI. White arrowheads show accumulation of DRiPs inside nucleoli, as previously published [12]. Scale bar is 10 μm. C Quantification of the number of SUMO1 nuclear foci in cells from A. Nuclear foci were segmented using the SUMO1 signal. Number of nuclei analyzed: 676 (siRNA control); 546 (siRNA PML); 546 (siRNA Ubc9). The average number of SUMO1 nuclear foci/nucleus is shown, +/− s.e.m. One-way ANOVA followed by Post-hoc Bonferroni-Holm test. D Quantification of the enrichment of DRiPs inside SGs in cells from (A). SGs were segmented using the TIA1 signal. Number of SGs segmented and quantified in three independent experiments: 3708 (siRNA control); 5710 (siRNA PML); 4833 (siRNA Ubc9). SGs with DRiPs’ enrichment > 1.5 is shown, +/− s.e.m. One-way ANOVA followed by Post-hoc Bonferroni-Holm test.
