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Figure 4 | Molecular Cancer

Figure 4

From: Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

Figure 4

H2AX leads to a redistribution of the transcriptional machinery. (A) Immunofluorescence analysis of U-2 OS cells for expression of pRNA pol II S2 (Covance, Berkeley, CA) 24 h after transfection with empty GFP vector (control), GFP-H2AX or myc-H2A. Note chromatin aggregation and the speckled pRNA Pol II S2 pattern in cells transfected with H2AX. Nuclei stained with DAPI. Scale bar indicates 10 μm. (B,C) Quantification of U-2 OS cells with redistributed or lost RNA pol II S2 expression 24 h after transfection with empty GFP vector (control in B), GFP-H2AX, GFP-H2AX-S139A, empty myc-tag vector (control in C) or myc-H2A. Each bar indicates mean + standard error of at least three independent experiments with at least 100 cells counted per experiment. (D) Double-immunofluorescence analysis of U-2 OS cells for expression of pRNA pol II S2 and CBP (Santa Cruz, Santa Cruz, CA) 24 h after transfection with empty GFP vector (control) or GFP-H2AX. Note the severely reduced expression of pRNA pol II S2 and CBP in the H2AX-transfected cell. Scale bar indicates 10 μm. (E) Quantification of U-2 OS cells with reduced CBP expression 24 h after transfection with empty GFP vector (control), GFP-H2AX, or GFP-H2AX-S139A. Each bar indicates mean + standard error of at least three independent experiments with at least 100 cells counted per experiment. (F) Double-immunofluorescence analysis of U-2 OS cells for expression of pRNA pol II S2 and p300 24 h after transfection with empty GFP vector (control) or GFP-H2AX. Note the severely reduced expression of pRNA pol II and p300 in H2AX-expressing cells. Scale bar indicates 10 μm. (G) Immunoblot analysis of pRNA pol II S2 in soluble nuclear (S3) and chromatin bound (P3) protein fractions obtained from U-2 OS cells transiently transfected (24 h) with empty GFP vector, GFP-H2AX or GFP-H2AX-S139A and treated with 10 μM aphidicolin (APH) for 1 h to block DNA replication.

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