Fig. 2

Overexpression of Api5 in MCF10A cells alter acinar morphology with increased proliferation. A Representative image of day 16 acini showing nuclei stained with Hoechst 33258 (blue) and mCherry (green). Violin plot showing (B) surface area and (C) volume of day 16 acini measured using Huygens software (SVI, Hilversum, Netherlands). D Violin plot showing the number of cells in each acinus, manually counted in day 16 Hoechst 33258-stained acini. Statistical analysis was performed using the Mann–Whitney test. E Bar diagram showing the percentage of acini with partially or entirely filled lumen. Day 16 acini stained with Hoechst 33258 were analysed manually by Huygens software. F Lysates collected from Api5 OE day 16 acini were immunoblotted for Ki67. G Quantification showing fold change in Ki67 protein level normalised to GAPDH (Samples from same experiment processed in 2 parallel gels as GAPDH is not detectable on 6% gel due to lower molecular weight). Statistical analysis was performed using the paired t-test. H Representative image of day 16 acini immunostained for Ki67 (green) and nuclei with Hoechst 33258 (blue). I The percentage of acini with more than 33% Ki67 positive nuclei were manually analysed and plotted as a bar graph. Statistical analysis was performed using an unpaired t-test. J Protein expression of PCNA in day 16 acinar cultures was obtained using western blot and quantified (K). Statistical analysis was performed using the paired t-test. L Representative image showing colony formed on soft agar assay and represented as a bar graph showing the total number of colonies formed. Statistical analysis was performed using the Mann–Whitney test. Control and Api5 OE cells were sparsely seeded and tracked for 3 h to analyse (M) distance travelled (N) velocity, (O) persistence and (P) displacement. Q Representative data showing movement of single cells in control and Api5 OE grown as monolayer cultures, sparsely seeded and tracked for 3 h. Statistical analysis was performed using the Mann–Whitney test. Data was pooled from N ≥ 3 independent experiments. For all statistical tests, *P < 0.05, **P < 0.01, ***P < 0.001and ****P < 0.0001. Full