Figure 5.

Oncosphere generation is dependent on the ATP-generating activities of NUDT5. (A) Bright field images of oncospheres formed following 5 days in culture in NUDT5^RES cell lines grown in the absence or presence of 1.5 nM TH5427. Scale bar represents 200 μM. (B) Quantification of the area of oncospheres formed in NUDT5^RES cell lines in the presence or absence of TH5427; mean ± SEM. (C) Quantification of the number of oncospheres formed in NUDT5^RES cell lines in the presence or absence of TH5427; mean ± SEM. (D) Stable knock-down and inducible pKAR rescue experimental overview in T47D^M cells. (E) Relative mRNA levels of NUDT5 following the addition of doxycycline to NUDT5^RES cell lines. (F) Bright field images of oncospheres formed following 5 days in culture in NUDT5^RES cell lines grown in the presence of doxycycline for 24 h after seeding. Scale bar represents 200 μM. (G) Quantification of the area of oncospheres formed in NUDT5^RES cell lines in the presence or absence of doxycycline, 24 h after seeding. (H) Immunofluorescent staining of phosphor-T45 NUDT5 in 2D and 3D cell culture conditions. (I) Bright field images of oncospheres formed following 5 days in culture in NUDT5^TD cell lines. (J) Quantification of second generation self-renewal capacity in NUDT5^RES (± TH5427), NUDT5^KD and NUDT5^TD cell lines, five days after primary 3D culture disassociation; data represents mean ± SEM. (K) qRT-PCR gene expression analysis of CSC marker; ALDH and EMT marker; EGFR1 and tumour marker; MUCIN1 in NUDT5^RES (±TH5427), NUDT5^KD and NUDT5^TD cell lines; data represents mean ± SEM.