Figure 1.
Effect of SIRT1 suppression on telomerase activity in human cells. (A) Suppression of SIRT1 in BJ-hTERT cells (BJT). BJT cells were infected with a control shRNA expressing retrovirus pSRP-shControl or SIRT1 knockdown virus pSRP-shSIRT1(HS6). Cell lysates were subjected to immunoblotting using an anti-SIRT1 antibody or a β-actin antibody. (B) Suppression of SIRT1 in Lovo cells. The same retroviral vectors as in A and in analysis were used. (C) Suppression of SIRT1 in Hela cells. The same retroviral constructs as in A were used. (D) Suppression of SIRT1 in Hela cells using control shRNA retrovirus and two shSIRT1 (HS6) and shSIRT1 (HS11) constructs.(E) Parental BJ cells expressing ectopic pM (MSCV)-hTERT-IEGFP were infected with pSRP-shControl RNA or pSRP-shSIRT1 (HS6) virus. Within 6 PDs after selection in puromycin, cells were lysed in CHAPS lysis buffer, and equal protein quantities (50 and 300 ng) were subjected to TRAP analysis to determine telomerase activity. Control RNase and heat treatments all contained 300 ng of protein lysate. (F) Lovo cells were infected twice with the viruses and were subjected to TRAP analysis. Protein amounts of 50, 200, and 600 ng were used in the TRAP reaction. (G) Same as F except Hela cells were used. (H) Same as in G except internal controls were included using HeLa cell extracts (10, 50, and 200 ng). (I) A second shRNA (HS11) was used to suppress SIRT1 in Hela cells and TRAP analysis was performed as in H. (J) Same cells as in A (BJ-hTERT-IEGFP with and without shRNA against SIRT1) were infected with pBabe-neo (PBN) control vector or with an shRNA-resistant silent SIRT1-R expressing construct (PBN-SIRT1-R) generating four additional lines. Two protein concentrations (50 and 200 ng) from four cell line were subjected (total of 16) to the TRAP analysis. The first six lanes are experimental controls. Lanes 7 and 8 are results of rescue experiments. The last three lanes are negative controls for the TRAP reaction.