American Society of Hematology

Figure 5

$Figure 5. SIRT6 plays multiple roles in the DDR of MM cells. (A) Cell viability assays of control and SIRT6KD MM cells following DDA treatment, with or without U0126 (10µM) or AS703026 (100 µM). Viable fraction is expressed as a percentage of the viability values obtained for the respective untreated conditions. (B) Survival curves of control and SIRT6-KD MM.1S cells upon DDAs treatment in the presence or absence of the specific RSK inhibitor fmk (1-3 μM). Results represent 3 experiments; error bars denote SD. Survival histogram of MM.1S SIRT6KD cells transfected with nontargeting (ctr) or RSK2-targeting siRNAs (3 individual clones) and treated for 48 hours with doxorubicin (100 nM) or melphalan (3 μM) (right). Survival fraction is expressed as percentage of the viability values obtained for the respective untreated conditions (mean ± SD). **0.003 < P < .001, ***P = .0009, ****0.0008 < P < .0001; ns, not significant (Student t test). Representative western blot (n = 3) shows RSK2 silencing 72 hours after transfection of MM.1S SIRT6-KD cells with 3 individual siRNAs targeting RSK2 (top). (C) ChIP analysis using a SIRT6-specific antibody to detect SIRT6 recruitment at Alu sequences after DNA damage triggered by doxorubicin (1 μM) or melphalan (100 μM), with or without pretreatment with OSS_128167 (200 μM). Occupancy at Alu sites is shown relative to background signal with IgG control antibody. n = 3 independent experiments. Data are presented as the mean ± SD. *P = .01, **P = .003 (Student t test) (D) ChIP analysis to detect H3K56 acetylation at DNA damage sites in SIRT6-WT and SIRT6-KD MM cells upon genotoxic stress. Antibody to acetylated H3K56 was used, and levels are shown relative to the background signal with IgG control antibody. n = 4 independent experiments. Data are presented as the mean ± SD. ****P < .0001 (unpaired t test). (E) Effect of SIRT6 depletion and overexpression on NHEJ and HR mechanisms. MM1S-overexpressing SIRT6-WT or SIRT6-depleted cells, as well as control cells, were cotransfected with NHEJ or HR reporter constructs and DsRed-encoding plasmids. The number of GFP+ and DsRed+ cells was determined by flow cytometry 72 hours later. The ratio of GFP+ to DsRed+ cells was used as a measure of repair efficiency. Representative fluorescence-activated cell sorter traces are shown (top). Data are shown as the mean values ± SD of triplicates. *0.03 < P < .02, **0.004 < P < .001 (Student t test). PCR, polymerase chain reaction.$

SIRT6 plays multiple roles in the DDR of MM cells. (A) Cell viability assays of control and SIRT6KD MM cells following DDA treatment, with or without U0126 (10µM) or AS703026 (100 µM). Viable fraction is expressed as a percentage of the viability values obtained for the respective untreated conditions. (B) Survival curves of control and SIRT6-KD MM.1S cells upon DDAs treatment in the presence or absence of the specific RSK inhibitor fmk (1-3 μM). Results represent 3 experiments; error bars denote SD. Survival histogram of MM.1S SIRT6KD cells transfected with nontargeting (ctr) or RSK2-targeting siRNAs (3 individual clones) and treated for 48 hours with doxorubicin (100 nM) or melphalan (3 μM) (right). Survival fraction is expressed as percentage of the viability values obtained for the respective untreated conditions (mean ± SD). **0.003 < P < .001, ***P = .0009, ****0.0008 < P < .0001; ns, not significant (Student t test). Representative western blot (n = 3) shows RSK2 silencing 72 hours after transfection of MM.1S SIRT6-KD cells with 3 individual siRNAs targeting RSK2 (top). (C) ChIP analysis using a SIRT6-specific antibody to detect SIRT6 recruitment at Alu sequences after DNA damage triggered by doxorubicin (1 μM) or melphalan (100 μM), with or without pretreatment with OSS_128167 (200 μM). Occupancy at Alu sites is shown relative to background signal with IgG control antibody. n = 3 independent experiments. Data are presented as the mean ± SD. *P = .01, **P = .003 (Student t test) (D) ChIP analysis to detect H3K56 acetylation at DNA damage sites in SIRT6-WT and SIRT6-KD MM cells upon genotoxic stress. Antibody to acetylated H3K56 was used, and levels are shown relative to the background signal with IgG control antibody. n = 4 independent experiments. Data are presented as the mean ± SD. ****P < .0001 (unpaired t test). (E) Effect of SIRT6 depletion and overexpression on NHEJ and HR mechanisms. MM1S-overexpressing SIRT6-WT or SIRT6-depleted cells, as well as control cells, were cotransfected with NHEJ or HR reporter constructs and DsRed-encoding plasmids. The number of GFP+ and DsRed+ cells was determined by flow cytometry 72 hours later. The ratio of GFP+ to DsRed+ cells was used as a measure of repair efficiency. Representative fluorescence-activated cell sorter traces are shown (top). Data are shown as the mean values ± SD of triplicates. *0.03 < P < .02, **0.004 < P < .001 (Student t test). PCR, polymerase chain reaction.

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