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. 2017 Jul 9;16(5):1094–1103. doi: 10.1111/acel.12639

Figure 4.

Figure 4

Akt and p21 are independently regulated in H‐Ras‐induced senescence. (A‐G) WI‐38 cells were infected with an H‐RasV12 retrovirus and treated with LY294002 from days 0 to 2 (LY 0–2 d). The experimental procedure for the induction of H‐Ras‐mediated senescence and LY294002 (LY) treatment (A). WI‐38 cells were harvested at the indicated time points and subjected to Western blot analysis using the indicated antibodies (B). SA‐β‐gal staining was examined at the indicated time points (C). Intracellular ROS levels were measured at the indicated time points using an EPICS XL cytometer following the staining of cells with DCF‐DA (50 μm) (D). A representative image of SA‐β‐gal staining captured on the 6th day is shown (E). Cell numbers were determined at the indicated time points by trypan blue exclusion (F). The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (G). (H–L) WI‐38 cells expressing either a p21 (shp21) or nontargeting (shNC) shRNA were infected with an H‐RasV12 retrovirus. SA‐β‐gal staining was performed at the indicated time points (H). Cell numbers were determined at the indicated time points by trypan blue exclusion (I). A representative image of SA‐β‐gal staining captured on the 6th day is shown (J). Intracellular ROS levels were measured at the indicated time points using an EPICS XL cytometer following the staining of cells with DCF‐DA (50 μm) (K). The percentage of cells in the S‐phase was examined using an EPICS XL cytometer (L). *P < 0.05; **P < 0.01 by Student's t‐test. NS, not significant.