![Figure 2. SIRT6 reduces MM cell proliferation by opposing MAPK-signaling pathway. (A) SIRT6 silencing using the lentiviral vector pGIPz (after 48 hours with 2 μg/ml puromycin selection) in MM.1S cells. Shown is a western blot for SIRT6 in SIRT6-silenced cells (with 3 different shRNAs) and scramble control. After 4 days of infection, cells were harvested and whole-cell lysates were subjected to immunoblot analysis. The indicated MM cell lines were infected with lentivirus coexpressing GFP and SIRT6 shRNA (clone #911) or scramble control (top). Western blot analyses were performed 4 days after transfection. Data are representative of 2 independent experiments (middle). The percentage of GFP+ cells was measured over time by flow cytometry and normalized to the percentage of scramble GFP+ cells. Data represent the mean values ± SD and represent a minimum of triplicates (bottom). (B) Colony formation of MM cells transduced with lentivirus expressing GFP-tagged scramble or shSIRT6. Light and florescence microscopy is shown. Numbers of colonies after 2 weeks (average of 3 independent experiments; unpaired t test; *0.05 < P < .04) (top). (C) Cell proliferation of MM.1S cells transduced with SIRT6-specific shRNA (clone #911), SIRT6 wild-type (WT) or specific controls was determined by [3H]- thymidine uptake. The results presented are a mean ± SD of triplicate samples (Student t test; *0.45 < P < .03; **0.006 < P < .001; ***0.009 < P < .006; ****P < .0001). (D) Representative western blots showing activation of MAPK-signaling pathway in MM cells depleted of SIRT6 compared with control. GAPDH was used as loading control. One representative blot of two is shown. (E) Representative western blots showing SIRT6 regulating ERK2 activity. MM.1S cells stably expressing shRNA-targeting SIRT6 were transduced with pLOC carrying SIRT6 open reading frame sequence lacking the 3′ UTR region (which is targeted by the siRNA), permitting rescue of the target-specific shRNA phenotype. (F) Western blots showing specific role of SIRT6 in MM.1S cells. One representative blot of two is shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/127/9/10.1182_blood-2015-06-649970/4/1138f2.jpeg?Expires=1735851215&Signature=TprHF-JLGifwipGfc2rtlW-SyHRKz0EBTQzaoUEhQWDTrzcBBUFxMOBTxFVIqJstIWRHHYNAkySvYw8LFfyDnM42B06b7jBAlRWvTXEq4mSvGcuImEbNkM2FBwNkRWZFQMHWqBtKf6C4M-Tk62w5ggu9DED9bcwK-MYelyZsj7z12tJ96F8MKOSM6iG8Xp74uf0WkSX-h5dw26Dmq1EgVFMRZyf6BomN92D9wTKDUFg7djdqaULB1sYK5EkeAqTJ7ycSEK07CQyiJrZ2-bINyykvzWXWCzkmdibi473tqipzbkb1cilmXWc62h0VuJ-ytEb1xFFBmuT8fmY0mS9QUA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
SIRT6 reduces MM cell proliferation by opposing MAPK-signaling pathway. (A) SIRT6 silencing using the lentiviral vector pGIPz (after 48 hours with 2 μg/ml puromycin selection) in MM.1S cells. Shown is a western blot for SIRT6 in SIRT6-silenced cells (with 3 different shRNAs) and scramble control. After 4 days of infection, cells were harvested and whole-cell lysates were subjected to immunoblot analysis. The indicated MM cell lines were infected with lentivirus coexpressing GFP and SIRT6 shRNA (clone #911) or scramble control (top). Western blot analyses were performed 4 days after transfection. Data are representative of 2 independent experiments (middle). The percentage of GFP+ cells was measured over time by flow cytometry and normalized to the percentage of scramble GFP+ cells. Data represent the mean values ± SD and represent a minimum of triplicates (bottom). (B) Colony formation of MM cells transduced with lentivirus expressing GFP-tagged scramble or shSIRT6. Light and florescence microscopy is shown. Numbers of colonies after 2 weeks (average of 3 independent experiments; unpaired t test; *0.05 < P < .04) (top). (C) Cell proliferation of MM.1S cells transduced with SIRT6-specific shRNA (clone #911), SIRT6 wild-type (WT) or specific controls was determined by [3H]- thymidine uptake. The results presented are a mean ± SD of triplicate samples (Student t test; *0.45 < P < .03; **0.006 < P < .001; ***0.009 < P < .006; ****P < .0001). (D) Representative western blots showing activation of MAPK-signaling pathway in MM cells depleted of SIRT6 compared with control. GAPDH was used as loading control. One representative blot of two is shown. (E) Representative western blots showing SIRT6 regulating ERK2 activity. MM.1S cells stably expressing shRNA-targeting SIRT6 were transduced with pLOC carrying SIRT6 open reading frame sequence lacking the 3′ UTR region (which is targeted by the siRNA), permitting rescue of the target-specific shRNA phenotype. (F) Western blots showing specific role of SIRT6 in MM.1S cells. One representative blot of two is shown.
