Fig 1: Nuclear CTSV trafficking is mediated via a bipartite nuclear localisation signal. (A) In silico analysis via NLS mapper has identified a potential bipartite NLS sequence motif (underlined residues) within CTSV. A CTSV expression construct with a mutated NLS was generated (mtNLS) by mutation of the basic lysine and arginine residues within the NLS motif to alanines (indicated in red). (B) Western blotting analysis following transfection of the mtNLS expression construct shows reduced nuclear CTSV protein expression in MCF-7 and ZR75-1 cells. (C) Confocal analysis of CTSV protein expression (red) following transfection of the mtNLS construct was used to confirm reduced nuclear CTSV expression. DAPI counterstaining (blue) was used to identify cell nuclei. Scale bars = 10 μm (D) MCF-7 and ZR75-1 cells were treated with ivermectin to confirm the functionality of the predicted NLS motif. Western blotting analysis of cytoplasmic (C) and nuclear (N) fractions confirmed nuclear localisation of CTSV was reduced following ivermectin treatment. HDAC1 and tubulin expression were included as loading controls relative to the nuclear and cytoplasmic fractions respectively and data presented is representative of three independent experiments.
Fig 2: CTSV is localized in the nuclear compartment of breast cancer cells. (A) Cytoplasmic (C) and nuclear (N) extractions from MCF-7 and ZR75-1 cells identified the presence of CTSV in both cellular compartments by Western blotting. (B) Confocal microscopy was undertaken to confirm the nuclear localisation of CTSV (red) with DAPI nuclear staining (blue). Scale bars = 10 μm (C) Cells sorting and Western blotting analysis suggested that CTSV is localised to the nucleus predominantly during S and G2/M phases. HDAC1 and tubulin expression were included as loading controls relative to the nuclear and cytoplasmic fractions respectively and data presented is representative of at least three independent experiments.
Fig 3: CTSV depletion attenuates growth of breast cancer cells by stalling progression through G2/M phase. (A) CTSV depletion in MCF-7 and ZR75-1 cells results in reduced cell growth when assessed by MTT assay. (B) PI staining with flow cytometry analysis identified that CTSV depletion results in fewer cells in G1, with a concomitant increase in G2/M phase. Rescue experiments were undertaken by restoring CTSV expression in sh1 cells, where cell cycle profiles were restored to that of the NTC cells. (C) Western blotting analysis of CTSV depleted cells shows that cyclin B1 expression is elevated in comparison to control cells, whereas cyclins D1/D2 and E1 remain unchanged. Tubulin expression was used as an internal loading control, with presented blots representative of at least three independent experiments. The average and standard deviation (SD) values are representative of three independent experiments, with statistical analysis was determined by two-way-ANOVA with a Tukey’s post hoc test for multiple comparisons performed using GraphPad Prism 8.
Fig 4: Histone H3 and H4 protein expression is regulated by CTSV. (A) Western blotting analysis of histone protein expression shows a reduction in histone H3 and H4 in CTSV depleted cells, with no impact on histones H1, H2a and H2b. (B) Confirmation of CTSV impact on histone H3 and H4 was ascertained using rescue experiments and the re-expression of CTSV in shCTSV-1 cells. GAPDH expression was used as an internal loading control and data presented is representative of at least three independent experiments.
Fig 5: Nuclear CTSV is required for histone H3, H4 and sNASP protein expression and cell cycle progression. (A) Restoration of wtCTSV (which traffics to the nucleus) expression in CTSV depleted cells (sh1) is necessary to reinstate histone H3, H4 and sNASP protein expression, while restoration from the mtNLS construct (which has impaired nuclear trafficking) is insufficient. (B) Confirmation of the impact of nuclear CTSV on cell cycle progression was observed by PI staining and flow cytometry. Restoration of CTSV from the wtCTSV construct was necessary to restore G1 and G2/M cell proportions to that observed in the NTC cells, restoration of CTSV from the nuclear deficient mtNLS construct had no impact on the cell cycle profile. Tubulin expression was used as an internal loading control, with presented blots representative of three independent experiments. The average and standard deviation (SD) values are representative of three independent experiments, with statistical analysis was determined by two-way-ANOVA with a Tukey’s post hoc test for multiple comparisons performed using GraphPad Prism 8.
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