Fig 2: Expression analysis of RAD51 and its paralogs in PTEN-mutated/deficient GBM. (A) Expression analysis of RAD51 and its paralogs stratified by the PTEN status from The Cancer Genome Atlas (TCGA) dataset. Box-and-whisker plots of log2-transformed gene expression levels in glioblastoma tissue specimens from TCGA sorted by the PTEN mutational status denoted as wild-type (WT; n = 95) or mutant/deletion (mut/del; n = 50). An unpaired Wilcoxon rank-sum test was conducted between PTEN WT and mut/del expression values for each gene, resulting in the displayed p values. (B) Expression analysis of RAD51 and its paralogs stratified by the PTEN status from patient-derived GBM brain tumor initiating cells (BTICs). Box-and-whisker plots of log2-transformed gene expression levels in BTICs from the SKS dataset sorted by the PTEN mutational status denoted as wild-type (WT; n = 7) or mutant/deletion (mut/del; n = 6). An unpaired Wilcoxon rank-sum test was conducted between PTEN WT and mut/del expression values for each gene, resulting in the displayed p values. (C) Western analysis of the BTICs derived from primary and recurrent GBM patients. Each BTIC line is derived from a GBM patient with an independent PTEN mutation (PTEN status) predicted to abrogate the PTEN protein stability/function based on the Gene Analysis Toolkit (GATK) sequence analysis (https://gatk.broadinstitute.org/hc/en-us; Broad Institute, Cambridge, MA, USA) and the previous studies [58]. RAD51 is expressed in all PTEN-mutant/deficient primary GBM BTICs, while the DNA damage generated by topotecan (TPT; 10 µM at 37 °C for 2 h) induces robust phosphorylation of 53BP1 (p-53BP1S1778) indicating active cellular non-homologous end joining (NHEJ) activity. The presence of phosphorylated ATM (p-ATMS1981) and KAP1 (p-KAP1S824) is indicative of induction of the DNA damage response. Irradiation of immortalized lymphoblastoid cells (LCLs) from normal (WT) and ataxia–telangiectasia (A–T) patients serve as ATM-proficient and -deficient control lines, respectively, to track ATM/KAP1-mediated DNA damage signaling.

Fig 3: TRIM28 negatively regulates necrosome-induced cytokine production. (A) mRNA expression levels of HT-29 cells expressing TRIM28 shRNA or the non-silencing control were analyzed by qPCR. (B) Cells from (A) were treated with TSZ for the indicated time points and analyzed by western blotting (left panel), LDH leakage (middle), and MTT assay (right panel). (C and D) Cells were treated with TSZ for indicated times. Their mRNA levels were analyzed by qPCR (C), and cell lysates were subjected to western blotting (D). (E and F) SNU-620 cells stably expressing TRIM28 shRNA or RIPK3 shRNA were treated with TSZ in a time-dependent manner. Cell lysates were analyzed by western blotting (E), and mRNA levels were analyzed by qPCR (F). (G) L929 cells were treated with TZ for 2 h, and IL-6 and CCL4 mRNA levels were analyzed by qPCR. (H) HT-29 cells expressing TRIM28 shRNA or a non-silencing control were fractionated into nuclear and cytosol fractions after the indicated treatments. Fractionation samples were analyzed by western blotting. (I) Representation of motifs enriched at TSZ-sensitive TRIM28 loss peaks (top) and TSZ-sensitive TRIM28 gain peaks (bottom). Known motif analysis was performed using HOMER; the top 10 ranked motifs are shown with their p-values. (J) RNA-seq-based expression of SOX family proteins in HT-29 cells (GSE108621). (K) Density plot of TRIM28 and SOX9 ChIP-seq datasets centered on 9412 TRIM28-occupied regions in HT-29 cells treated with DMSO or TSZ for 4 h. Each row represents a single peak. (L) Metagene representation of the ChIP-seq signal for SOX9 across 9412 TRIM28-occupied regions in HT-29 cells treated with TSZ for 4 h. Metagenes centered on the middle of 9412 regions and 10 Kb around the center of the TRIM28-occupied regions are displayed

Fig 4: Derepression of TRIM28 leads to increased synthesis of immunostimulatory cytokines. (A) Bone marrow-derived monocyte (BM) differentiated into dendritic cells (DC) upon culture with GM-CSF for 5 d. Conditioned medium from necroptotic cells (CM) or control cells was added for further activation. (B) DCs were treated with CM for 16 h, and CD86, CD40, and CD80 mRNA levels were analyzed by qPCR. (C) DCs were treated with CM for 16 h, and CD86 expression levels were analyzed by FACS. (D) DCs were treated with CM from L929 cells, which were treated with TZ for 120 min in the absence or presence of Nec-1 (CM), for 16 h. FACS analysis was performed. (E) DCs from RIPK3 WT or RIPK3 KO mice were treated with CM. CD86 expression was analyzed by FACS. (F and G) DCs were treated with CM from L929 cells stably expressing TRIM28 shRNA or the non-silencing control for 16 h. CD86 and MHC II expression was analyzed by FACS (F), and CD86, CD40 and CD80 mRNA levels were analyzed by qPCR (G). (H) The heatmap table shows the association between TRIM28 or RIPK3 and tumor-infiltrating level of multiple types of CD8+ T cell or DC, and that is estimated by six algorithms across cancer types in the TCGA database. Red indicates a statistically significant positive association, and blue indicates a statistically significant negative association. (I) Schematic of proposed RIPK3/TRIM28 activation-mediated immunostimulatory cytokine production

Fig 5: TRIM28 is the potential E3 ligase for SIRT1 and directly interacts with SIRT1. (A) Network view of proteins interacted with SIRT1 generated by inBio Discover, all the ubiquitination-related proteins are highlighted with blue circles. (B) Bar chart showing the potential E3 ubiquitin ligases for SIRT1 as predicted by the UbiBrower database. (C) HEK293T cells were transfected with FLAG-SIRT1 for 24 h and then treated with DMSO or DOX (5µM) for 8 h. Cell lysates were subjected to co-immunoprecipitation assay using anti-FLAG agarose and blotted with the relevant antibodies for western blot analyses. (D) HEK293T cells were transfected with FLAG- TRIM28 for 24 h and then treated with DMSO or DOX (5µM) for 8 h. Cell lysates were subjected to co-immunoprecipitation assay using anti-FLAG agarose and blotted with indicated antibodies after western blot analysis. (E) Diagrammatic representation of the truncated HA-TRIM28 plasmids structure. (F) HeLa cells were co-transfected with EGFP-SIRT1 and different HA-tagged TRIM28 truncating mutations as shown in (E) for 24 h and treated with DOX (5µM) for 8 h. Cell lysates were subjected to GFP IP and blotted with relevant antibodies.See also Figure S1.