SNX-2112 targets degradation of Hsp90 client protein. (A) The formal chemical structure of SNX-2112 and SNX-5422. (B) High content analysis images of various Hsp90 client proteins and phosphorylated proteins regulated by Hsp90 clients. Levels of p-ERK and Her2 were measured concurrently in proliferating AU565 cells using species specific fluorescein isothiocyanate- and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies, and images from each treatment condition are from identical cells. Exposure times for each channel were identical for all 3 treatment conditions to allow for direct comparison of staining intensities between treatment conditions. Levels of p-S6 were also measured concurrently in treated A375 cells. Levels of p-Akt were measured in serum-starved A375 cells activated with 100 ng/mL of IGF-1 after 24 hours of compound treatment. For measuring NF-κB translocation, HUVECs were activated with 1 ng/mL of IL-1β for 30 minutes after a 24-hour compound treatment, and nuclear staining intensity was quantified by segmenting individual nuclei based on Hoechst staining. For all images shown, individual cells were identified by Hoescht blue staining of nuclei. (C) Hsp90 client assay in MM cells. MM.1S cell were treated with SNX-2112 or 17-AAG for 24 hours at indicated dose, and the levels of Akt and IKBα protein were evaluated by Western blotting.