Fig 2: SMARCB1-NR4A3 interaction is enhanced by ROS. Determination of the effect of NAC or H2O2 on SMARCB1-NR4A3 interaction in (a) HUVEC or in (b) RAOEC under reoxygenation condition by the anti-SMARCB1 IP assay followed by western blot. Cells were subjected to 4-hour OGD treatment and reoxygenation for 1 hour (OGD-R). 4 mM NAC and 500 μM H2O2 were applied at the beginning of reoxygenation. For the co-IP assay, an isotype IgG was used to serve as a negative control.

Fig 3: Functional effects of sub-cellular location of truncated SMARCB1: Senescent cell formation (a) and induction of SA-β-gal activity (b), indicative of senescence by SMARCB1 and mutants. Upon transfection in MON (SMARCB1−/−) cells, SMARCB1 as well as SMARCB1(L266A) increases the percentage of senescent cells and SA-β-gal-positive cells, while SMARCB1(Q318X) (i.e., truncated protein shown to be of cytoplasmic location) does not induce senescent cells or SA-β-gal-positive cells. In contrast, disruption of the NES in SMARCB1(L266A;Q318X) double mutant (shown to restore nuclear location of truncated protein) significantly induces senescent cells that are positive for SA-β-gal staining. a The senescent cell images were captured at 20X using the phase contrast setting. b The cells were stained with SA-β-gal and the images were captured after 13 days at 20 × using the Zeiss Axio Observer CLEM (Correlative Light and Electron Microscopy). Each experiment was performed three independent times and a representative image per sample is shown. Panels in a and b represent images of MON cells transfected with: GFP (panel 1); GFP-SMARCB1 (Panel 2); GFP-SMARCB1(Q318X) (Panel 3); GFP-SMARCB1(L266A) (Panel 4); and GFP-SMARCB1(Q318X;L266A) (Panel 5). Panel 6 represents the Graphical representation of the quantitation of data using multiple sets of transfection experiments indicating % of senescent cells (a); or % SA-β-gal-positive cells (b). (mean ± SEM. ****p value < 0.0001, **p value < 0.01, ns not significant)

Fig 4: Effect of Selinexor (KPT-330) on cell growth and senescent cell formation in the presence and absence of SMARCB1 and SMARCB1(Q318X): Phase contrast microscopic visualization of senescent cell formation in MON (SMARCB1−/−) cells transfected with GFP, GFP-SMARCB1 or GFP-SMARCB1(Q318X) in response to Selinexor (KPT-330) 7 days (a) or 10 days (b) post-treatment. Images were captured at 20 × using the phase contrast setting. Shown are representative images. c and d Percentage of senescent cells per field of view of treated and untreated cells in a and b, Mean ± SEM. e–g Effect of Selinexor on cell survival. MON (SMARCB1−/−) cells transfected with GFP, GFP-SMARCB1 or GFP-SMARCB1(Q318X) were subjected to MTS cell proliferation assay at 0, 4, 7 and 10 days post-treatment with 100 or 500 nM Selinexor (% of treated compared to untreated, mean ± SEM)

Fig 5: Cytoplasmic SMARCB1 staining status according to SMARCB1 mutation. Immunohistochemical staining results using the BAF47 antibody in 49 ATRT, in which SMARCB1 SNVs/indels were encountered (a). Note that distinct cytoplasmic staining is highly over-represented in cases showing SNVs/indels C-terminal of the nuclear export sequence (NES). The majority of the SNVs/indels were nonsense (circles) and only one missense (square) and two intronic mutations (triangles) were encountered. # Missense mutation of the second allele (p.L43V), § Nonsense mutation of the second allele (p.Y47X). WHD Winged Helix domain; DBD DNA binding domain; RPT Repeat; NES Nuclear Export Signal; HR3 homology region 3 (coiled-coil domain). Representative staining examples for distinct (b), faint (c) as well as absent cytoplasmic SMARCB1 staining (d) are also given