Extended Data Fig. 6: PML recognizes and dissolves nuclear inclusions, and its overexpression promotes their clearance.
From: PML targets and resolves structured protein inclusions to mitigate neurodegeneration
a, Spatial relationship between PML localization and the polarity distribution of polyG inclusions. PML pseudocolor was adjusted for localization display with the polyG-FLIM image. CSLM, confocal scanning laser microscopy. b, Long-term live-cell imaging and cumulative intensity analysis of polyG–eGFP inclusions following recognition by mRuby3-PML, showing gradual dissolution of inclusions. Each arrowhead indicates a polyG inclusions. c, Schematic of pellet separation from HEK293T cells expressing polyG inclusions. PE., pellet fraction, SN., supernatant fraction. d, Fluorescence images of the pellet fraction from HEK293T cells expressing polyG–eGFP with or without PML–HA. e, Immunoprecipitation of eGFP and eGFP-tagged uN2C-polyG variants from HEK293T cells co-expressing PML–HA. Immunoblotting revealed that PML–HA specifically interacts with uN2C-polyG104×-eGFP. f, Immunoprecipitation of HA-tagged PML truncations from HEK293T cells co-expressing polyG–eGFP showing that the SRS2 domain of PML is required for its interaction with polyG–eGFP inclusions. g, Schematic indicating that PML-I lacks the complete SRS2 domain. h, Immunoblot of polyG–eGFP in lysate fractions from cells expressing PML-I or -IV variants showing PML-I exhibits reduced ability to eliminate polyG inclusions. i, Immunofluorescence (top) and quantification (bottom) of cells co-expressing polyG–eGFP and PML–HA (For intensity analysis, n = 15 coated and 20 diffused polyG aggregates were analysed; for proportion analysis, 8 fields from two independent experiments were measured). A.U., arbitrary unit. j, Fluorescence images (left) and quantification (right) of polyG–eGFP inclusions in cells expressing HA or PML–HA with or without MG132 treatment (10 μM, 6 h) (mean ± s.d.; n = 4 fields from two independent experiments per group). k, Schematic of WT PML and the SUMO E3 ligase-deficient mutant PML-M6 (top) and immunoblot of polyG–eGFP in lysate fractions from cells expressing PML-WT-HA or PML-M6-HA (bottom). l, Coomassie staining of purified PMLΔCC–GST protein. m, Microscale thermophoresis (MST) analysis showing that PMLΔCC binds polyG aggregates with a dissociation constant (Kd) of 23.8 μM (mean ± s.d.; n = 4 independent experiments). n, Thioflavin T (ThT) binding assay of preassembled polyG aggregates (25 μM, 37 °C, 24 h) treated with PMLΔCC–GST (mean ± s.d.; n = 3 independent experiments). o, Turbidity analysis of preassembled polyG aggregates (25 μM, 37 °C, 24 h) treated with 10 μM PMLΔCC–GST, monitored by optical density at 300 nm (blue) and 600 nm (red) (mean ± s.d.; n = 3 independent experiments). p, Representative fluorescence images of HEK293T cells expressing polyGA–eGFP together with HA or PML–HA at the indicated ratios. q, Quantification of the number and total area of polyGA inclusions shown in p (mean ± s.e.m.; n = 8 fields from two independent experiments per group). r, Fluorescence images of the pellet fraction from HEK293T cells expressing polyGA–eGFP with HA or PML–HA. s, Immunoblot of polyGA–eGFP in lysate fractions from HEK293T cells expressing HA or PML–HA. t, Representative immunofluorescence images of PSMB1 and DnaJB1 in cells co-expressing polyGA–eGFP and PML–HA. Line profiles show fluorescence intensities along the dashed arrows. u, Representative immunofluorescence images of HEK293T cells expressing polyGA–eGFP with or without PML–HA, followed by treatment with or without cytoskeleton-stripping buffer, which selectively removed polyGA–eGFP within PML–HA condensates. All statistical significance was determined using two-tailed unpaired Student’s t-test.
