Dual inhibition of AKT‐mTOR and AR signaling by targeting HDAC3 in PTEN‐ or SPOP‐mutated prostate cancer

A

LNCaP cells were treated with 10 ng of IGF‐1 for 30 m and harvested for immunofluorescent cytochemistry (IFC) with P‐AKT‐S473, E‐cadherin, and HDAC3. Cell nuclei were counterstained with DAPI in IFC. Scale bars, 50 μm.

B

C4‐2 cells were transfected with the indicated plasmids, and then, cytoplasm membrane and nuclear proteins were isolated followed by IP and Western blots with the indicated antibodies.

C

An illustration depicts functional domains of HDAC3 including the regions for nuclear export and localization sequences (modified from the following website: http://atlasgeneticsoncology.org/Genes/GC_HDAC3.html).

D, E

LNCaP cells were transfected with HA‐tagged wild‐type HDAC3 or truncated mutant (aa 1–313) for 24 h followed by IFC with anti‐HA antibody (D) or Western blots with indicated antibodies (E). Scale bars for images in (D), 50 μm.

F

C4‐2 cells were transfected with a pool of siRNA of APLL1 and the indicated plasmids. The cells were harvested for IP and Western blots with the indicated antibodies.

Figure EV2. The C‐terminal region (aa 1–313) in HDAC3 is indispensable for its cytoplasm exportation that is a prerequisite for AKT phosphorylation and AR upregulation.