SIRT6 inhibition sensitizes MM cells to doxorubicin treatment in vivo. (A) Whole-body imaging of CB17-SCID mice bearing GFP-expressing NCI-H929 scramble (scr) or SIRT6-KD stably transduced cells treated with vehicle or doxorubicin. (B) Growth of NCI-H929 control and SIRT6-depleted xenografts in mice treated with vehicle or doxorubicin (doxo) (P = .011 and P = .013, respectively). Data are mean tumor volume ± SD. SIRT6 was measured by western blot in representative tumors harvested from mice on day 30. Mean RTV ± SEM (n = 4) for mice treated in panel A on days 16 and 30 is expressed compared with tumor volumes on day 1 (insert). (C) Kaplan-Meier analysis showing median survival times of mice bearing tumors with or without SIRT6, before and after treatment with vehicle or doxorubicin. (D) Immunohistochemical analysis for hematoxylin and eosin (H&E), γH2AX, and RAD51 focus formation in control and SIRT6-KD NCI-H929 xenografts harvested from mice treated with either vehicle or doxorubicin. Black arrows indicate γH2AX nuclear foci. (E) Tumor sections from treated and untreated mice were subjected to immunostaining with anti-GFP and anti-γH2AX. Nuclear staining was performed with DAPI. For panels, Original magnification ×20 (D-E) (×40 in insets). (F) Mean tumor growth assessments showed smaller tumor sizes in mice with SIRT6-KD xenografts than in other groups (left). RTV for mice treated on days 16 and 30 (right). (G) Kaplan-Meier survival plot showing median survival of mice bearing conditional SIRT6-KD xenografts with doxycycline diet and treated with vehicle or doxorubicin (left). Western blot of harvested tumors confirms SIRT6 silencing in doxycycline-fed mice, as well as increased γH2AX after treatment (right). Tx, therapy.