Figure 1.
INMAP overexpression suppressed the growth of HeLa cells.
(A, B). Colony formation of HeLa cells in monolayer cultures. Cells were stained with 10% Giemsa stain. Data are presented as mean ± SD; n = 4. ** and * represent significant differences with P-values under 0.01 and 0.05, respectively (the same below). NS, not significant. (C, D). Anchorage-independent colony growth assay in soft agar. Cells were seeded in 12-well plates containing 0.3% soft agar. After 6 weeks, the number of colonies with more than 50 cells was counted. Frequency of colonies of HeLa cells stably overexpressing INMAP in soft agar was analysed. Scale bars, 50 μm.
Figure 2.
INMAP overexpression inhibited tumour growth in vivo.
(A). Control (HeLa and Flag-HeLa) and overexpressing INMAP (Flag-INMAP) cells were injected into Balb/c nude mice (n = 5) in the back (near right axillary). 40 d later, the tumour xenografts were stripped. (B-D). Average tumour weights and sizes and mouse body weights were determined, respectively. No significant differences were detected. (E). Western blot analysis of the tumour tissues with anti-PCNA, anti-Bcl-2 antibodies. GAPDH was used as an internal control. (F). The histopathological analysis of the tumour tissues from different groups with H&E staining. Scale bars, 20 μm (a, c, e) and 5 μm (b, d, f). Larger necrotic areas were found in INMAP-overexpressing tumour tissue. (G). Morphological characteristics of normal (a) and abnormal (b) livers in nude mice. Inflammatory areas of the liver are labelled with arrowheads. (H). The histopathological analysis of normal (a, b) and abnormal (c, d) livers. Multiple necrotic areas, strongly stained nuclei, and infiltrated neutrophils were observed in abnormal livers. Scale bars, 20 μm (a, c) and 5 μm (b, d).
Figure 3.
INMAP overexpression induces genomic instability in HeLa cells.
(A, B). The effect of INMAP overexpression on the induction of micronuclei. (A). Mono-, bi- and tri-nucleated cells, stained with Giemsa. Arrowhead indicates micronucleus. (B). The frequency of micronucleated cells in Flag-INMAP cells (n = 400) is greater than in Flag-HeLa or HeLa. (C). The numbers of chromosomes were determined in INMAP-overexpressing cells (n = 100). Scale bars, 5 μm.
Figure 4.
INMAP overexpression induces DNA damage in HeLa cells.
(A). Western blot analysis of HeLa, Flag-HeLa and Flag-INMAP cells with anti-γH2AX and anti-GAPDH antibodies. (B). Each band quantification was analysed with Image J software. Data are representative of three independent experiments. AU, arbitrary unit.
Figure 5.
(A). Colocalisation of INMAP and p21 in INMAP overexpression HeLa cells (Flag-INMAP). Cells were double-stained with mouse monoclonal anti-Flag antibody (green) and rabbit monoclonal anti-p21 antibody (red), and stained cells were analysed with IIF. Where green and red signals overlap (Merge), a yellow pattern is observed, indicating the colocalisation of INMAP and p21. Scale bars, 5 μm. (B). Pulldown assay of INMAP and p21. Expression vector pET30a-INMAP was transformed into Escherichia coli BL21. The purified His-INMAP fusion protein was added into HeLa cell extracts, incubated, and recovered on beads. pET30a (+) was a control. The Pulldown product was analysed by Western blotting. (C, D). CoIP assay of INMAP with p21. The proteins of HeLa cells that expressed Flag-INMAP were extracted and immunoprecipitated with the monoclonal Flag antibody (C) or p21 antibody (D). The same amount of purified mouse IgG (C) or rabbit IgG (D) was used in control samples. The immunoprecipitation (IP) result was analysed with SDS-PAGE and Western blot with anti-p21 or anti-Flag antibody.
Figure 6.
Effects of INMAP overexpression on the expression of genes related to proliferation and apoptosis.
(A). Western blot analysis of the three groups of cells (HeLa, Flag-HeLa, and Flag-INMAP) with anti-Bcl-2, anti-p53, anti-p21 and anti-PCNA antibodies. Data shown are representative of three independent experiments. (B). Band quantification was analysed with Image J software. (C). A schematic model illustrating the proposed mechanisms of INMAP-overexpression-induced DNA damage and apoptosis through p53-dependent pathways in transformed cells.