Fig 2: Pterostilbene significantly inhibits MTA1-dependent cell proliferation and angiogenesis and induces MTA1-targeted apoptosis in Ptenf/f mice(A) Representative Ki-67 (top, each panel), CD31 (middle, each panel) and VEGF-C (bottom, each panel) staining of the prostate tissues from Ptenf/f mice treated with vehicle and PTER, at indicated ages. Arrows indicate vessels. Scale bars, 100 µm. (B) Quantitation of Ki-67 (top) and CD31 (bottom) positive cells of prostate tissues from mice treated with vehicle and PTER (n = 5/group). (C) Representative images and (D) Quantitation of cleaved Caspase-3 staining at the indicated ages of vehicle and PTER treated Ptenf/f mice (n = 5/group). Scale bars, 10 µm. Data are mean ± SEM (n = 3), *p < 0.05; **p < 0.01; ***p < 0.001 (two-tailed, two-sample t-test). (E) Immunoblots of MTA1, Ac-p53, p53 and Bak in the prostate tissues from vehicle and PTER treated Ptenf/f mice, isolated at the indicated ages. NP, normal prostate. Hsp70 was used as loading controls from prostate tissues. (F) Densitometry of the Ac-p53/p53 ratio from the representative blot. (G) Comparative analysis of MTA1 binding in the prostate tissues of Pten+/f mice on Ctrl- and PTER-Diet. Representative MTA1 ChIP-Seq tracks for Vegf-c gene locus at 10 kb resolution are shown.

Fig 3: Identification of DNAJC9 as a dual histone chaperone and heat shock co-chaperone(A) Schematic representations of the histone H3-H4 (red and cyan, respectively) binding mode of MCM2 (dark green) and TONSL (yellow) highlighting histone binding mutants (Huang et al., 2015; Saredi et al., 2016) and the experimental strategy for the identification of histone-dependent interactors. Hypothetical histone-dependent interactors (colored light green and orange for MCM2 and TONSL, respectively) and histone-independent interactors (gray) are depicted.(B and C) Mass spectrometry analysis of SILAC labeled pull-downs of wild-type (WT) and histone binding mutant (HBM) forms of MCM2 (B) and TONSL (C) from soluble cell extracts; n = 2 biological replicates. Proteins referred to by human UniProt protein identification code. See also Table S1.(D) Pull-downs of full-length GST-DNAJC9 mixed with pre-assembled H3.3-H4 (top) and MCM2 HBD-H3.3-H4 complexes (bottom).(E) Pull-downs of FLAGHA-tagged histone H3 variants induced by doxycycline (Dox+) compared with control purifications (Dox-) from soluble cell extracts. Western blots representative of n = 2 biological replicates.(F) Plasmid supercoiling assay showing histone chaperone activity of DNAJC9 compared with ASF1A and NASP HBD as positive controls. R, relaxed DNA; S, supercoiled DNA.(G) Schematics depicting the ability of the DNAJC9 N-terminal J domain to stimulate HSP70 catalysis (Han et al., 2007) and the histone-dependent interactions formed between DNAJC9, MCM2, and TONSL.

Fig 4: Caspase-dependent HLJ1 reduction in apoptotic cells. (A) Decreased expression of HLJ1 was pronounced upon inhibition of protein synthesis. CL1-0 cells were pre-incubated with 10 µg/ml of CHX for an hour, and exposed to 10 J/m2 of UV. After recovery for 6 h, HLJ1 expression was analyzed by western blot using anti-HLJ1 antibody (left) and real-time RT–PCR (right). (B) The majority of detached cells induced by UV irradiation were apoptotic. CL1-0 cells were irradiated with 10 J/m2 of UV, and allowed to recover for 24 h. Based on adherence, total cells including detached and adherent, adherent cells and detached cells, were harvested. The DNA content of cells was analyzed by flow cytometry, as described in ‘Materials and Methods’ section. (C) HLJ1 was absent in detached cells. CL1-0 and HeLa cells were treated with 10 J/m2 of UV irradiation, and allowed to recover for 24 h. Total cells (T), adherent cells (A) and detached cells (D) were collected for western blot analysis with anti-HLJ1 and anti-Hsc70 antibodies. ß-Tubulin was applied as the internal control. Results are representative of three independent experiments. (D) Caspase inhibitors attenuated UV-induced cell apoptosis. CL1-0 cells were pre-incubated for 3 h with 20 µM zVAD-fmk or 30 µM DEVD-fmk, followed by exposure to 10 J/m2 of UV irradiation. After recovery for 6 h, the apoptotic cell content was determined based on the sub-G1 proportion using a flow cytometer. Data are presented as mean ± S.D. (n = 3). (E) Caspase inhibitors blocked UV-induced HLJ1 degradation. Western blots were performed with antibodies against HLJ1, PARP or ß-tubulin. PARP is a caspase-3 substrate, while ß-tubulin is the internal control.

Fig 5: BCL-6 aggregate formation upon co-expression of exogenous HSP70 and HSP90.ECs were transfected with EFp-BCL-6 in combination with HSP70-1A or HSP90-HA expression plasmid or with EFp-Link control vector. After 12 and 24 hours cells were stained for CLSM imaging with antibodies against BCL-6 and HSP70 or with antibodies against BCL-6 and HA-tag for HSP90 detection. Hoechst 33342 was applied to counterstain nuclear DNA. (A) CLSM images demonstrate the concomitant overexpression of BCL-6 with HSP70 or HSP90 at 24 hours after transfection (scale bars: 10 µM). (B) The percentage of BCL-6 positive cells was determined from four randomly acquired tile scans (40x objective) covering approximately 700 cells. Mean values and standard deviations from two independent experiments are given; T-test revealed no statistically significant differences. (C) 35 randomly selected nuclei with BCL-6 aggregations were analyzed (per condition) for the diameter of their aggregates. The boxplot illustrates the distribution of acquired values with a statistically significant reduction in aggregate diameter upon co-expression of HSP70 after 12 h (T-test; p < 0.001) and 24 h (T-test; p = 0.001). (D) 300 BCL-6 positive cells (per condition) were classified according to their nuclear BCL-6 expression pattern into diffuse (no aggregation), small punctate, large spherical and additional cytosolic aggregates. Mean values and standard deviations were calculated from 2 independent experiments. The frequency of large, spherical aggregates was significantly reduced upon co-expression of BCL-6 and HSP70 after 24 h (T-test; p = 0.031). *, p < 0.05; **, p < 0.01; ***, p < 0.001.