Figure 6. USP28 is required to maintain CSC-like features in breast cancer cells via LSD1.
(A) Phase-contrast micrograph of MMTV-Wnt1 cells stably transfected with control vector, USP28 shRNA or in a USP28-knockdown clone with LSD1-rescued expression. Scale bar = 100 μm.
(B) Tumorsphere-formation is examined in cells above. Representative images of tumorsphere were shown. Scale bar = 100 μm.
(C) Quantification of tumorspheres from cells in (B) was plotted and shown.
(D) CD49flow/CD24high population in cells above was analyzed by FACS.
(E) Representative images of the 3D structures formed by cells above in Matrigel (top panel) were presented. Cellular structures were stained for CK14 and E-cadherin (bottom panel; nuclei were stained with Dapi). Scale bar = 20 μm.
(F) Data presented are a quantification of the numbers of solid organoid and acini-forming structures from (E).
(G) mRNA levels of several basal and luminal genes from cells above were quantified by real-time RT-PCR. Values were normalized to 18S.
(H) Lysates from cells above were analyzed by Western blotting.
In panel C, D, F, and G, data presented are representative of three experiments performed in duplicates as the mean ± SD. *p value < 0.05; **p value < 0.01; and ***p value < 0.001 when the control group or rescued group was compared with two individual clones expressing USP28 shRNAs.