Fig 1.
Ki-67 expression in non-cancerous and cancerous cells upon serum starvation.
Confocal analysis of Ki-67 expression in HDF, HUVEC, MDA-MB-231, HeLa and FaDu cells (analysed over time after serum deprivation), and the relative fluorescence signal quantification (average overlap value percent). (Two-way ANOVA, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Scale bar: 10 μm.
Fig 2.
Ki-67 gene knock-down experiment.
(A) Confocal analysis of Ki-67 expression in HDF and MDA-MB-231 cells after shRNA knock-down. Bottom part: graph showing the Ki-67 protein quantification. (B) RT-qPCR quantification of the α and β splice variants of Ki-67, and GAPDH (G), after virus knock-down. The green graph represents the internal control GAPDH. For both cell models the relative quantification of GAPDH internal control (Ctrl) during Ki-67 gene knockdown experiments is also reported. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001). Scale bar: 15 μm.
Fig 3.
Simple Western™, Standard Western, and quantification profile of Ki-67 in (A) HDF and (B) MDA-MB-231 breast cancer cells, growing in both media supplemented (+FBS) and lacking of FBS (-FBS). In the figure it is also reported the Simple Western™ quantification profile of the internal control ERK 1/2 in HDF and MDA-MB-231 breast cancer cells, before and after FBS deprivation. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).
Fig 4.
RT-PCR (left column), and RT-qPCR (right column) quantification of the α and β splice variants of Ki-67, as a function of nutrient starvation in HDF and MDA-MB-231 cells. Bottom figure: (left) RT-PCR analysis of β-actin (A) and GAPDH (B) as internal controls, before and after nutrient deprivation. (Right) RT-qPCR quantification of the internal control GAPDH after serum starvation. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).
Fig 5.
Ki-67 and proteasome interaction.
Confocal assays and quantification graphs (on the bottom of the picture) of the distribution and interaction between Ki-67 and the proteasome system, in both HDF (left column) and MDA-MB-231 cells (right column). The analyses were carried out with and without the presence of FBS. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).
Fig 6.
Ki-67 extranuclear distribution.
Confocal assays of the distribution and interaction between Ki-67 and BiP (A), COPII (B) and Golgi (C), in HDF (left column) and MDA-MB-231 cells (right column), before and after serum deprivation. The quantification graphs (on the left and right sides of the picture) show the overlap signal between Ki-67 and the complex of interest, for HDF and MDA-MB-231 growing in both media supplemented and lacking of FBS. For a single channel analysis, see Figures D-G of S1 File. (t-test, p-value * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001).
Fig 7.
Proposed molecular regulation and extranuclear pathway of Ki-67.
Scheme representing the proposed Ki-67 regulation in non-cancerous (HDF) and cancerous cells (MDA-MB-231) before and after serum deprivation. In non-cancerous cells (left column), prolonged serum deprivation produces the down-expression of both the α and β splice variants of Ki-67. Furthermore, the already expressed Ki-67 is degraded via the proteasome. On the other hand, cancerous cells (right column), subjected at the same starving conditions, show the continuous expression of the α splice variant of the protein. Moreover, the level of the detected Ki-67 in cancerous cells is not affected overtime by the degradative action of the proteasome. The “feedback elimination mechanism” of Ki-67 degradation, which is related to the ER-Golgi secretory machinery, is schematised in the box at the bottom of the figure. In this proposed mechanism, Ki-67 is initially transferred to the ER, where it colocalises with BiP (1). Subsequently, Ki-67 buds from the ER into specific COPII coated vesicles (2), which transport their cargo to the Golgi apparatus (3) where Ki-67 may be further recycled and/or degraded. Non-cancer and cancer cells translocate Ki-67 with the same mechanism. However the obtained data indicate that cancerous cells could be characterised by a defective ER-Golgi secretory machinery. (4) Moreover, the Ki-67 translocation in cancerous cells could be unbalanced by the non down-regulation of the α variant of the protein.