Figure 6: eLIM increases cancer cell radiosensitivity via mitotic catastrophe and apoptosis. | Nature Communications

Figure 6: eLIM increases cancer cell radiosensitivity via mitotic catastrophe and apoptosis.

From: A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity

Figure 6

(a) HeLa cells were treated with the indicated peptides (50 nM), irradiated (8 Gy) and analysed for the proportion of mitotic cells by FACS as in Fig. 4a. Representative FACS and immunoblot with the indicated antibodies are shown. CHK2i, CHK2 inhibitor II (100 nM). (b) Clonogenic survival assays of HeLa cells treated as in a. (c) Immunofluorescence analysis of HeLa cells treated with the indicated peptides or CHK2i and irradiated. Cells were stained with anti-α-tubulin (green) and anti-γ-tubulin (red). The nuclei were stained with DAPI (blue). If green and red colours overlap, yellow colour appears, and if red colour overlaps with blue colour, purple appears. Yellow arrows indicate cells with multipolar spindles and white arrows indicate cells with polymorphologic nucleus. The percentages of aberrant mitotic cells were plotted. Scale bars, 100 μm. (d) Representative FACS analysis of apoptosis stained with Annexin V and PI in HeLa cells treated as in c. Data shown are mean±s.d. of three independent experiments. *P<0.05, **P<0.01 versus no peptide without IR, #P<0.05, ##P<0.01 versus no peptide with IR (a–d). (e) Volume of xenograft tumours derived from HeLa cells treated with the indicated peptide or CHK2i as indicated. The different number (1–10) refers to different mice. Data are shown as mean±s.d. (n=10) (*P<0.05, **P<0.01 at 28 days). Representative tumour tissues (No. 5) were subjected to immunoblot analysis with the indicated antibodies. The P values were generated using two-tailed Student’s t-test (a–e). (f) Proposed model for LIM proteins-mediated modulation of the G2/M checkpoint and radioresistance. IR induces the expression of LIM proteins (For instance, IR stimulates FHL1 transcription in a SP1- and MLL1-dependent manner). LIM proteins increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequesters CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, leading to inactivation of the CDC2/cyclin B complex and subsequent G2/M arrest. eLIM blocks these processes, resulting in mitotic catastrophe and apoptosis.

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