Fig. 8.

Cell-penetrating MH2 protein could be a potential treatment for glioma. a Schematic representation of the TAT-MH2-NLS construct (upper). TAT-MH2-NLS protein was expressed in 293T cells and purified as described in the Methods section (lower). b Nuclear accumulation of TAT-MH2-NLS protein. 293T cells were treated with 0.5 μg ml⁻¹ TAT-MH2-NLS for 6 h before fixation. Immunofluorescence (IF) staining was performed with anti-His (red) and anti-β-tubulin (Tubulin, green) antibodies and cell nuclei were stained with Hoechst (Hoe, blue). c TAT-MH2-NLS protein bound to PIAS3. 293T cells were transfected with HA-PIAS3 and treated with 1.0 μg ml⁻¹ TAT-MH2-NLS and followed by IP analysis with anti-His antibody. d TAT-MH2-NLS inhibited Smad6-induced PIAS3 ubiquitination and degradation. 293T cells were transfected with indicated constructs and incubated with indicated dose of TAT-MH2-NLS protein. After 24 h treatment, cells were treated with 10 μM MG132 for 4 h before collecting. IP was performed using anti-HA antibody followed by IB with indicated antibodies. e TAT-MH2-NLS protein antagonized STAT3-mediated transcriptional activation. Luciferase assay of SIE activity was performed in HEK293 cells transfected with HA-PIAS3 and treated with indicated amounts of TAT-MH2-NLS (n = 3). f TAT-MH2-NLS increased endogenous PIAS3 expression in U87 and U251 cells. g, h TAT-MH2-NLS inhibits GBM tumor growth. The tumor-bearing mice were treated with TAT-NLS (Con) or TAT-MH2-NLS (MH2). Bioluminescent images (g) and quantification (h) of xenografts derived from U87 implantation (n = 10). i TAT-MH2-NLS treatment prolongs the animal survival. Kaplan–Meier survival curves of mice implanted with U87 cells (Log-rank χ² = 10.88, P < 0.001. n = 10). Data were represented as means ± SD and analyzed using two-tailed Student’s t-test in e, h. *P < 0.05, **P < 0.01