(A and B) Schematic diagram of (A) and protein expression/activity in (B), the HMEC transformation model.

(C) Relative levels of aggresomes in matrix-attached and -detached HMEC, PHMLEB, and PHMLER.

(D and J) Reduced K48 polyUb-modified proteins during HMEC transformation (D) and in breast cancer cells (J). NS, NP-40 soluble; SS, NP-40 insoluble and SDS soluble. Relative ratios of K48 polyUb in the SS versus total (NS + SS) fractions were quantitated based on three independent experiments (n = 3; SD = ± 4%).

35S]Met-labeled proteins in the SS (DRiPs) fractions versus total labeled proteins (NS + SS) are shown (n = 3; SD = ±2%). The representative images are shown in (E and K) Increased clearance of DRiPs during HMEC transformation (E) and in breast cancer cells (K). Relative ratios of [S]Met-labeled proteins in the SS (DRiPs) fractions versus total labeled proteins (NS + SS) are shown (n = 3; SD = ±2%). The representative images are shown in Figures S1 D and S1H.

(F and L) Levels of FLAG-Atxn1 82Q stably expressed in the indicated cells, and relative Atxn1 82Q/GAPDH ratios (n = 3; SD = ±3%).

(G) Representative images of cells stably expressing Atxn1 82Q-GFP (green) and stained with DAPI (blue). Scale bar, 50 μm. (H and M) Stability of FLAG-Atxn1 82Q analyzed by cycloheximide (CHX) chase. Relative Atxn1 82Q/GAPDH ratios are shown (n = 3; SD = ±2%).

(I) Stability of FLAG-Atxn1 82Q analyzed by pulse chase. Atxn1 82Q was immunoprecipitated (IP). Levels of [35S]-labeled Atxn1 82Q relative to time 0 are indicated (n = 3; SD = ±2%).

(N) Schematic diagram of ATRA-induced NB4 differentiation.

(O and P) Morphology of (O; scale bar, 50 μm), and relative levels of aggresomes in (P), NB4 cells treated with vehicle (DMSO) or ATRA (1 μM).

(Q) Levels of GFP-Atxn1 82Q, GFP-Atxn1 30Q, and GFP stably expressed in DMSO- or ATRA-treated NB4 cells.

(R and S) Expression (R) and stability (S) of Atxn1 82Q-GFP expressed in DMSO- and ATRA-treated NB4 cells were examined by fluorescence microscope (R; scale bar, 50 μm) and CHX chase (S), respectively.

In (C), (E), (K), and (P), data represent mean or mean ± SEM (n = 3). ∗∗p < 0.01; ∗∗∗p < 0.001.