Figure 2

TRIP12 is a cell cycle-regulated nuclear protein associated with chromatin. (A) TRIP12 protein level in cytosolic and nuclear fractions of HelaS3 cells was measured by Western blot analysis. SP1 and 4EBP1 protein levels were used as purity controls of subcellular fractions. Images were obtained from the same experiment and representative of three different experiments. (B) TRIP12 subcellular localization in HelaS3 cells was determined by immunofluorescence using TRIP12 antibody (Sigma). The inset represents TRIP12 nuclear localization at a higher magnification. Nuclei were counterstained with DAPI. The white arrows indicate the peri-nucleolar and peri-nuclear heterochromatin regions. (C) TRIP12 nuclear expression in HelaS3 cells in the different phases of the cell cycle was determined by immunofluorescence using TRIP12 antibody (Sigma). Nuclei were counterstained with DAPI. Cells in G₁ phase and G₂ phase correspond to CYCLIN A/EDU nuclear negative cells and CYCLIN A nuclear positive/EDU negative cells, respectively. Cells in early G₁ correspond to G₁ cells with small oblong-shaped nucleus. Cells in early S and late S phase correspond to EDU positive cells/CYCLIN A negative and positive nuclear cells, respectively. The graph represents the mean TRIP12 expression (integrated density-background) ± SEM determined at least 200 cells using FIJI software. (D) Level of TRIP12 in cytosolic and nuclear protein fraction of G₁-, early S- and G₂-phase enriched HelaS3 cell populations was measured by Western blot analysis. SP1 and 4EBP1 protein levels were used as purity control of subcellular fractions. Images were obtained from the same experiment and representative of three different experiments. (E) TRIP12 and γ-TUBULIN localization in HelaS3 cells in prophase, prometaphase, metaphase, anaphase, telophase and cytokinesis were visualized by immunofluorescence. Nuclei were counterstained with DAPI. (F) TRIP12 expression in soluble protein (SP) and chromatin-bound (Chro) protein fractions (40 µg for each fraction) obtained from nocodazole-treated mitotic HelaS3 cells was measured by Western blot analysis. As mitotic cells do not have nuclear membrane, SP fraction contains cytoplasmic and nuclear soluble proteins. SP1, 4EBP1 and panH3 protein levels were used as purity control of the different fractions. Images were obtained from the same experiment. (G) TRIP12 localization on metaphasic chromosomes was visualized by immunofluorescence using anti-TRIP12 antibody (Sigma) after chromosome spreading of HelaS3 cells treated with Ro-3306 and released in the cell cycle for 45 min. DNA was counterstained with DAPI. The inset represents a magnification of TRIP12 localization on two individual chromosomes.