Fig. 5

Api5 knock-down in malignant breast cells resulted in a partial reversal of cancerous phenotypes. A Api5 protein expression in MCF10A cell line series. B Quantification showing the expression levels of Api5 across the MCF10A cell line series normalised to loading control GAPDH. Statistical analysis was performed using the paired t-test. C Api5 KD and control MCF10CA1a cells cultured on Matrigel® for seven days stained with nuclear stain Hoechst 33258 (blue). MCF10CA1a control and MCF10CA1a Api5 KD spheroids were stained with phalloidin, and morphometric analysis was performed to calculate (D) surface area and (E) volume of spheroids using Huygens software (SVI, Hilversum, Netherlands). Statistical analysis was performed using the Mann–Whitney test. F The percentage of cells with > 100 or < 100 cells per spheroids were calculated and plotted. G Percentage of spheroids with more than 50% Ki67 positive cells per spheroid. Statistical analysis was performed using an unpaired t-test. H Immunoblotting of lysates collected from day 7 3D culture probed for PCNA. I Plot showing the total number colonies formed on soft agar. Statistical analysis was performed using the Mann–Whitney test. J Representative image of tumour dissected from flanks of athymic mice, 8 weeks post subcutaneous injection. The left panel shows MCF10CA1a control cells, while the right panel shows Api5 KD MCF10CA1a (Number of mice:—6). K Graph showing tumour area as measured using vernier calliper at the mentioned time. Statistical analysis was performed using 2-way ANOVA followed by Sidak’s multiple comparisons test. Data are pooled from n > 5 independent experiments. L Api5 KD and control MCF10AT1 cells were cultured on Matrigel.® for 7 days and stained with Hoechst 33258. Morphometric analysis was performed on MCF10AT1 control and Api5 KD spheroids stained with phalloidin and (M) surface area and (N) volume of spheroid were measured using Huygens software. Statistical analysis was performed using the Mann–Whitney test. O The number of cells per spheroids was counted based on nuclear staining and plotted for the percentage of spheroids with > 100 (red) and < 100 cells (green). Statistical analysis was performed using an unpaired t-test. Data pooled from N ≥ 3 independent experiments. For all statistical tests, *P < 0.05, **P < 0.01, ***P < 0.001and ****P < 0.0001