Figure 2: ABRO1 suppresses cell growth in a p53-dependent manner.
From: ABRO1 suppresses tumourigenesis and regulates the DNA damage response by stabilizing p53
(a) Cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and 2 × 103 cells were plated per 6-cm dish and selected with G418. Clone numbers (>50 cells) were determined after 2 weeks. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. *P<0.05, **P<0.01. (b) HCT116 p53+/+ and HCT116 p53−/− cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and the number of G418-resistant clones was determined. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3a. (c) HCT116 p53+/+ and HCT116p53−/− cells were infected with control lentivirus, with lentivirus encoding shRNA specific to ABRO1 or with lentivirus encoding ABRO1 shRNA and RNAi-immune ABRO1. Clones were counted 2 weeks after initial plating. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3b. (d) L02 cells were infected with control lentivirus, with lentivirus encoding shRNA specific to ABRO1 or with lentivirus encoding ABRO1 shRNA and RNAi-immune ABRO1, and plated on soft agar. Clones were counted 2 weeks after initial plating. The data shown are the means±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. A representative image of the clones is shown in Supplementary Fig. 3c. (e) HCT116 p53+/+ and HCT116 p53−/− cells were transfected with pCMV-Flag-ABRO1 or pCMV-Flag vectors, and G418-resistant cells were selected. The cells were pulsed for 1 h with BrdU before being harvested, fixed, stained with anti-BrdU fluorescein isothiocyanate (FITC) and propidium iodide (PI), sorted by flow cytometry and analysed for DNA content. The data shown are the mean±s.d. of three independent experiments and were analysed using Student’s t-test. **P<0.01. (f) Volume of xenograft tumours derived from control or ABRO1-overexpressing HCT116 cells. n=10 in each group. Tumour sizes were measured using a calliper at the indicated time points. The data shown are means±s.d. (g) Excised tumours from control or ABRO1-overexpressing HCT116 cells. Scale bar, 10 mm.
