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. Author manuscript; available in PMC: 2017 May 19.
Published in final edited form as: Mol Cell. 2016 May 12;62(4):479–490. doi: 10.1016/j.molcel.2016.04.011

Figure 5. Clinical Relevance and Demonstration that U2AF35(S34F)-transformed Ba/F3 Cells have an Autophagy Defect, Mitochondrial Dysfunction and an Increased Spontaneous Mutation Frequency.

Figure 5

(A) Immunoblot monitoring LC3B-I, LC3B-II and p62 in Ba/F3-V, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA cells.

(B) Fluorescent Cyto-ID assay. Ba/F3-V, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA cells were treated with or without PP242 and stained with Cyto-ID, a fluorescent dye that selectively labels autophagic vacuoles.

(C) Proliferation of Ba/F3-V cells (cultured in the presence of IL-3) or Ba/F3-U2AF35(S34F) cells (cultured in the absence of IL-3) cultured in the presence or absence of doxycycline. (D and E) FACS analysis of Ba/F3-V, Ba/F3-U23AF35, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA cells cultured in the presence of IL-3 and double-stained with MitoTracker Green and MitoTracker Red

(D) or stained with MitoSOX

(E). The MitoTracker Green-positive, MitoTracker Red-negative population is encircled; quantification of this population is provided in the lower left-hand corner of the plot.

(F) Spontaneous mutation frequency in Ba/F3-V, Ba/F3-U2AF35, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA control (water) or ascorbate-treated cells, as measured by resistance to 6-thioguanine (6-TG; left) or ouabain (right). The results were normalized to that obtained in control Ba/F3-V or Ba/F3-NSshRNA cells, which was set to 1.

(G) Proliferation of Ba/F3-V, Ba/F3-U2AF35, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA cells treated with etoposide or, as a positive control, the autophagy inhibitor 3-methyladenine (3-MA), or both.

(H) Proliferation of Ba/F3-V, Ba/F3-U2AF35, Ba/F3-U2AF35(S34F), Ba/F3-NSshRNA or Ba/F3-Atg7shRNA cells treated with x-ray irradiation, or x-ray irradiation and 3-MA. Error bars indicate SD. *P<0.05; **P<0.01. See also Figure S5.