Fig 1: Cell cycle and proliferation of HeLa cells with irradiation-induced ING4 expression. (A) Percentage of G1/S and G2/M phase HeLa cells with stably integrated EGR1 promoter and ING4 open reading frame irradiated by X-rays. Compared with those of HeLa cells without irradiation, higher percentages of cells in G2/M phase and lower percentages of cells in G1/S phase were observed in HeLa cells treated with 4, 6 and 8 Gy irradiation. **P<0.001 vs. cell percentage without irradiation (0 Gy). (B) Distribution of phases of HeLa cells with or without inducible ING4 expression. ING4 expression significantly increased the percentage of cells in G2/M phase and decreased the percentage of cells in G1/S phase. **P=0.008 vs. without EGR1 promoter. (C) The photo of colony formation assay of HeLa cells with and without inducible ING4 expression under irradiation. With increasing irradiation, decreased numbers of colonies were observed for both groups. (D) Comparison of the colony numbers between ING4-expressing HeLa cells and non-inducible cells. Once ING4 was induced, less colonies were observed in HeLa cells. **P<0.001 vs. cell number without irradiation (0 Gy). EGR1, early growth response factor-1; ING4, inhibitor of growth 4.
Fig 2: Cell cycle analyses of HeLa cells by flow cytometry under the control of different doses of irradiation. The cell counts of HeLa cells stably integrated with EGR1 promoter and ING4 ORF, irradiated by (A) 0, (B) 2, (C) 4, (D) 6 and (E) 8 Gy, in G1/S and G2/M phase. (F) The cell counts of HeLa cells stably integrated with ING4 ORF without irradiation-induced EGR1 promoter, and irradiated by 8 Gy, in G1/S and G2/M phase. Events, cell counts; M1, G1/S phase; M2, G2/M phase; EGR1, early growth response factor-1; ING4, inhibitor of growth 4; ORF, open reading frame.
Fig 3: Apoptosis analysis of HeLa cells by flow cytometry with irradiation under hypoxia. Percentages of sub-G1 phase HeLa cells stably integrated with ING4 ORF without irradiation-induced EGR1 promoter, irradiated and analyzed at (A) 0, (B) 24, (C) 48 and (D) 72 h post-irradiation. Percentages of sub-G1 phase HeLa cells stably integrated with EGR1 promoter and ING4 ORF, irradiated and analyzed at (E) 0, (F) 24, (G) 48 and (H) 72 h post-irradiation. M1, percentages of sub-G1 phase cells (apoptotic cells); EGR1, early growth response factor-1; ING4, inhibitor of growth 4; ORF, open reading frame.
Fig 4: Irradiation-activated EGR1 promoter drives mCherry and ING4 expression in stably integrated HeLa cells. (A) HeLa cells were stably integrated with EGR1 promoter and mCherry ORF. mCherry expression increased due to the positively regulated promoter activity under irradiation (upper right panel). Magnification, ×100. (B) ING4 expression was detected by western blotting. HeLa cells stably integrated with ING4 ORF without the EGR1 promoter irradiated by different doses of X-ray exhibited ING4 expression was hardly visible. (C) ING4 expression was monitored by western blotting. HeLa cells stably integrated with EGR1 promoter and ING4 ORF exhibited increased ING4 expression following treatment with a gradient of X-rays. EGR1, early growth response factor-1; ING4, inhibitor of growth 4; ORF, open reading frame.
Fig 5: ING4 inhibits HIF-1a expression and promotes cell apoptosis. (A) Inducible ING4 expression and its effect on HIF-1a expression. When treated with CoCl2-imitated hypoxia, HeLa cells exhibited high expression levels of HIF-1a which were inhibited by ING4 induced by irradiation. (B) Effect of ING4 on HeLa cell apoptosis in CoCl2-imitated hypoxia under 8 Gy irradiation. ING4 expression significantly promoted the apoptosis of HeLa cells at 48 and 72 h compared with cells transfected with 0-ING4 (without EGR1 promoter), **P<0.05.. CoCl2, cobalt chloride; EGR1, early growth response factor-1; HIF-1a, hypoxia-inducible factor 1a; ING4, inhibitor of growth 4.
Supplier Page from Abcam for Anti-ING4 antibody