Figure 2.

Overexpression of Spi1 and HRAS^V12 in Lin⁻Kit⁺Sca1⁺ (LSK) cells leads to senescence. (A) Western blot analysis of Spi1, HRAS^V12 and the senescence marker Dec1 in hematopoietic cells subjected to the retroviral-mediated expression of Spi1, HRAS^V12 or an empty vector. Protein extracts of GFP-positive sorted cells were analyzed 7 days post-infection as described in Online Supplementary Figure S2. α-adaptin served as the loading control. (B) Number of total living cells retrovirally transduced with Spi1 and HRAS^V12 or an empty vector (control), at the indicated periods of time. The means ± SEM of at least 3 independent experiments are shown. (C) Representative SA-βgal staining and mean percentages of SA-βgal positive cells (histograms) in samples of hematopoietic cells subjected to the retroviral-mediated expression of Spi1, HRAS^V12 or an empty vector as a control. SA-βgal assays for sorted GFP-positive cells were performed 7 days post-infection as described in Online Supplementary Figure S2. The counting of GFP-positive SA-βgal cells was performed in 9 randomly selected fields with a total of more than 2000 cells from each group. Magnification of images, 200X. The means ± SD of at least 3 independent experiments are shown. **P<0.005; ***P<0.0005 from two-tailed Student’s t-test. (D) Flow cytometric detection of SA-βgal activity using C₁₂RG as a fluorogenic substrate in cells retrovirally transduced with Spi1, HRAS^V12 or an empty vector. The histograms represents the % of C₁₂RG positive cells among GFP-positive cells. The means ± SD of at least 3 independent experiments are shown. **P<0.005 from two-tailed Student’s t-test.