BMP signaling and SMURF1 are differentially regulated in CD44high/ALDHhighsorted CSC-like populations. Representative FACS analyses of sorted cells are shown in A and B. (A) ALDEFLUOR stained cells in the presence of ALDH activity inhibitor DEAB and double-stained with APC-conjugated IgG isotype control. (B) Gating of CD44low/ALDHlow and CD44high/ALDHhigh populations. (C–D) Immunoblotting of SMURF1, pSMAD1/5/8, and SMAD1 (as a control for total SMAD protein) in the sorted TR146 (C) and SCC-58 (D) cells shown as representatives of at least three repeats from two independent cell sorting experiments. CD44+ cells in the CD44low/ALDHlow sorted population were magnetically captured and removed to generate CD44depleted/ALDHlow cells. β-actin was used as a total protein loading control for all samples.