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Fig. 7 | Journal of Experimental & Clinical Cancer Research

Fig. 7

From: Upregulation of ERK-EGR1-heparanase axis by HDAC inhibitors provides targets for rational therapeutic intervention in synovial sarcoma

Fig. 7

Heparanase promotes histone acetylation and its inhibition reduces nuclear localization. a Serum starved CME-1 cells were incubated with human active recombinant heparanase (5 μg/ml) for the indicated times. Then, cells were lysed and processed for immunoblotting with the specified antibodies to detect heparanase (r, recombinant 50 kDa heparanase; e, endogenous 65 kDa heparanase) and acetylation of H3 (K27) and H4 (K12). Histone acetylation was also analyzed in cell lysates after transfection with aspecific RNA oligonucleotide (NegCTR) or HPSE siRNA for 72 h (b) and in cells treated with OGT2115 (0.5 μM) for 48 h or SST0001 (0.5 mg/ml) for 24 h (c). Vinculin, GAPDH and tubulin are shown as controls for protein loading. d Indirect immunofluorescence showing localization of heparanase in control and SST0001-treated (1 mg/ml for 18 h) cells. Nuclei are evidenced with Hoechst 3341 counterstaining (blue). Original magnification, 1000X. e Cytoplasmic and nuclear fractions from cells exposed to 0.5 mg/ml SST0001 or 1.6 μM SAHA for 18 h, were analyzed by Western blotting to examine intracellular distribution of heparanase polypeptides. Lamin B and GAPDH are shown as controls for nuclear-cytoplasmic fractioning and loading. f Biotin-conjugated SST0001 analogue SST0762NA1 enters the nuclei. Serum starved cells treated with SST0762NA1 (1 mg/ml) for 24 h were fixed, permeabilized, and incubated with streptavidin Alexa Fluor 488 conjugate to detect the drug. Cells were stained with Hoechst 33341 to evidence nuclei. Inset, enlarged detail evidencing SST0762NA1 localization in the nucleus and in perinuclear vesicles

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