Fig 2: TIF-IA controls the abundances of nuclear factor Y and DREAM complex transcriptional targets.(a,b) NHDFs were transfected with non-silencing (ns) control siRNA or a TIF-IA siRNA. Three days post transfection total RNA was isolated and RNA-seq was performed and (a) a volcano plot showing differentially expressed genes identified from RNA-seq of cells treated with TIF-IA siRNA (adjusted p-value<0.01) was generated and (b) Gene ontology analysis (GOTERM_BP_DIRECT) of genes downregulated more than four-fold in TIF-IA depleted cells compared to ns siRNA treated cells was performed using DAVID Functional Annotation Clustering Tool (n = 2). (c) TRANSFAC analysis of genes downregulated more than log(2) in TIF-IA depleted cells compared to ns siRNA treated cells was performed using GATHER to identify common modes of transcriptional regulation. (d) 3 d post-transfection of NHDFs with ns or TIF-IA targeting siRNAs, total protein was collected, separated by SDS-PAGE, and analyzed by immunoblotting using antibodies specific for p53 and Akt (loading control) (n = 3). (e) 3 d post-transfection of NHDFs with ns or TIF-IA targeting siRNAs, total RNA was isolated and RT-qPCR was performed for HMGB2 mRNA, CENPF mRNA, CDK1 mRNA, TK1 mRNA and CDCA8 mRNA. Error bars indicate SEM. *p≤0.05; **p≤0.01; Student's t test (n = 3). (f) As in d using the indicated siRNAs and measuring HMGB2 mRNA levels.

Fig 3: Cytoplasmic HMGB2 abundance is controlled by TIF-IA and dictates the cellular responses to dsDNA.(a) 3 d post-transfection of NHDFs with non-silencing (ns) control or TIF-IA targeting siRNAs, protein was collected, separated on an SDS-PAGE gel, and analyzed by immunoblotting using antibodies specific for TIF-IA, HMGB2, and actin (loading control) (n = 2). (b) NHDFs were transfected with ns, TIF-IA, or HMGB2 siRNAs. 3 d post-transfection cells were fixed in 4% PFA and immunofluorescence staining was performed using an antibody specific for HMGB2 (n = 2). Nuclei were visualized following DAPI staining. (c–e) NHDFs were transfected with ns or HMGB2 siRNA and (c) infected with HCMV, (d) infected with VSV, or (e) treated with cGAMP and (c–e) 6 hr later, total RNA was isolated, and RT- qPCR was performed using primers specific for IFNB1 mRNA. Error bars indicate SEM. *p≤0.05; Student's t test (n = 3). (f) model depicting how ribosome biogenesis regulates cellular responses to dsDNA via HMGB2. (Left panel) Normal rDNA transcription precludes nucleolar stress-induced p53 stabilization, permitting HMGB2 transcription by RNA pol II in the nucleoplasm and HMGB2 protein (shown in red) accumulation in the cytoplasm. Upon detecting non-microbial or HCMV cytoplasmic dsDNA, HMGB2 either alone or together with a dsDNA sensor like cGAS stimulates IFNB1 mRNA accumulation. (Right panel) Inhibiting RNAPI by TIF-IA depletion results in nucleolar stress and stabilizes p53, which in turn represses HMGB2 transcription, reduces HMGB2 protein abundance and restricts IFNB1 transcription in response to dsDNA.

Fig 4: NK-EVs cleave granzyme A substrates SET and HMG2 proteins in the target cells.Neuroblastoma CHLA255 (A) or ALL SupB15 (B) cells were treated with different amounts of NK-EVs (upper panel) or harvested at different time-points after treatment (lower panel) as indicated. Untreated cells were used as controls (CL). After incubation, cells were harvested and lysed in SDS-PAGE sample buffer. Each lane contains 30-μg cell lysate proteins as determined by the Orbit® protein assay (Life Technologies). Antibodies against SET (Santa Cruz Biotech, sc-133,138) were used to probe the blots. The same blot was stripped and re-probed with anti-HMG2 antibody (Cell Signalling Technology, #14,163). β-Actin was used as the loading control. Western blot signals were qualified by the program ImageJ from at least three independent experiments (n ≥ 3). The plots with the standard derivation were shown in Supplementary Figure S5.

Fig 5: Proteins identified by proteomics analysis of B cell MVs are detectable in both cell lysates and MVs of isotype and anti-CD24 stimulated B cells by Western blot analysis. Cell lysates (5 µg; equivalent to approximately 2.3 × 105 cells) and the corresponding protein from MVs (from 1.0 × 106 cells) were analyzed for expression of SHMT2, EEF1G, GRB2 and HMGB2. HSP90 was used as a loading control after stimulation with isotype (Iso) or anti-CD24 (CD24). n = 5. Two different outcomes were observed for HMGB2, with MVs from CD24 stimulated cells containing high HMGB2 (Upper panels, n = 2) or HMGB2 being low/absent in all vesicles (lower panels n = 3).