Article Figures & Data
Figures
- Figure 1. Knockdown of DAO inhibits DNA damage–induced senescence.
(A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 7 d, and the expression levels of DAO were determined by qPCR. (B) HepG2 cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 10 μM etoposide for 48 h, and the expression levels of DAO were determined by immunoblot analysis. (C, D) U2OS (C) and HepG2 (D) cells depleted of DAO were treated with 2 and 10 μM etoposide for 7 d and 48 h, respectively, and subjected to SA-β-gal staining. The percentage of SA-β-gal–positive cells (C left panel, D) and representative microscopic images (C right panel) are shown. Bars, 50 μm. (E) U2OS cells depleted of DAO were treated with 2 μM etoposide for 7 d and subjected to colony-formation assay. Relative proliferation rate (upper panel) and representative images (lower panel) are shown. (F) U2OS cells treated as in (E) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. Data are mean ± SD (n = 3 except in (A) where n = 2 independent experiments). Statistical significance is shown using the t test analysis; *P < 0.05, **P < 0.01.
- Figure 2. Pharmacological inhibition of DAO impairs DNA damage– and oncogene-induced senescence.
(A–D) U2OS (A, C) and HepG2 (B, D) cells treated with etoposide in the presence of 50 or 100 μM CBIO were subjected to SA-β-gal staining (A, B) and colony-formation assay (C, D). (E, F) U2OS (E) and HepG2 (F) cells were treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO, and the expression levels of the indicated proteins were determined by immunoblot analysis. The protein levels relative to the γ-tubulin levels, except for the phosphorylated p53 (p53 pS15), which was normalized to the total p53 levels, were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (G, H) Hs68 cells treated with 0.5 μM etoposide for 7 d in the presence of 50 μM CBIO were subjected to SA-β-gal staining (G) and EdU incorporation assay (H). The percentage of EdU-positive cells (H, left panel) and representative microscopic images (H right panel) are shown. Bars, 50 μm. (I) Hs68 cells treated with 0.5 μM etoposide for 2 d in the presence of 50 μM CBIO were subjected to immunoblot analysis. The protein levels were quantified as in (E, F). (J) WI-38 cells were transfected with pcDNA3-HA containing oncogenic RasG12V, and the protein expression of RasG12V was confirmed by immunoblot analysis. (K, L) WI-38 cells transfected as in (J) were selected with 300 μg/ml G418 and treated with 50 μM CBIO. After incubation for 8 d, the cells were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). Data are mean ± SD (n = 3 independent experiments). Statistical significance is shown using the t test analysis; **P < 0.01.
- Figure 3. Ectopic expression of wt-DAO, but not the inactive mutant, promotes senescence.
(A) U2OS cells were transfected with pcDNA3-HA containing wt- and R199W-DAO, and the protein expression of DAO was confirmed by immunoblot analysis. (B, C) U2OS cells transfected as in (A) were selected with 800 μg/ml G418 and treated with 2 μM etoposide. After incubation for 7 d, the cells were subjected to SA-β-gal staining (B) and EdU incorporation assay (C). Data are mean ± SD (n = 3 independent experiments). Statistical significance is shown using the t test analysis; *P < 0.05, **P < 0.01.
- Figure 4. d-arginine and d-serine enhances the senescence-promoting effect of DAO.
(A, B) U2OS cells were transfected with pcDNA3-HA containing wt-DAO, selected with 800 μg/ml G418, and treated with each of seven d-amino acids at 5 mM (except for d-tyrosine which was used at 2.5 mM) in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (A) and EdU incorporation assay (B). (C, D) U2OS cells were transfected as in (A, B) in the presence of d-arginine, d-serine, l-arginine, or l-serine at 5 mM for 7 d and subjected to SA-β-gal staining (C) and EdU incorporation assay (D). Data are mean ± SD (n = 3 independent experiments). Statistical significance is shown using the t test analysis; *P < 0.05, **P < 0.01.
- Figure 5. Riboflavin treatment activates the DAO activity in the absence of DNA damage.
(A) U2OS cells transfected with siRNA for RFVT1 were treated with 2 μM etoposide for 7 d, and the expression levels of RFVT1 were determined by qPCR. (B) U2OS cells depleted of RFVT1 were treated with 2 μM etoposide for 7 d and subjected to FAD quantification. The concentrations of FAD per mg protein of the cells are shown. (C, D) U2OS cells were transfected with pcDNA3-HA containing wt-DAO, selected with 800 μg/ml G418, and treated with 50 μM riboflavin and 5 mM d-serine in the presence of 2 μM etoposide as indicated. After incubation for 7 d, the cells were subjected to SA-β-gal staining (C) and EdU incorporation assay (D). (E) U2OS cells were treated as in (C, D), and the expression levels of IL-6 were determined by qPCR. (F) U2OS cells overexpressing DAO were treated with 50 μM riboflavin and 5 mM d-serine for 7 d and subjected to immunostaining for 53BP1, HA, and Hoechst staining. Representative microscopic images (left) and box plots of the number of 53BP1 foci in HA-DAO–expressing cells (right) are shown. The upper and lower limits of the boxes and the lines across the boxes indicate the 75th and 25th percentiles and the median, respectively. Error bars (whiskers) indicate the 90th and 10th percentiles, respectively. Statistical significance (P-value) is shown using the t test analysis (n = 50 cells). Data are mean ± SD (n = 3 independent experiments). Statistical significance is shown using the t test analysis; *P < 0.05, **P < 0.01, and n.s.: not significant (P > 0.05).
- Figure 6. DAO enhances DNA damage–induced ROS accumulation, cooperating with PRODH.
(A) U2OS cells transfected with siRNAs for DAO (DAO-1 and DAO-2) were treated with 2 μM etoposide for 2 d, and the ROS level was measured. (B, C) U2OS (B) and HepG2 (C) cells treated with 2 and 10 μM etoposide, respectively, for 2 d in the presence of 50 μM CBIO were subjected to ROS assay. (D) U2OS cells transfected with pcDNA3-HA-DAO and treated with 2 μM etoposide were subjected to ROS assay. (E, F) U2OS cells overexpressing DAO were treated with etoposide in the presence of 1 mM NAC for 7 d and subjected to SA-β-gal staining (E) and EdU incorporation assay (F). (G) U2OS cells treated as in (E, F) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels relative to the γ-tubulin levels were quantified using NIH ImageJ software and are indicated at the bottom of each lane. (H, I) U2OS cells treated with etoposide in combination with 50 μM CBIO and 1 mM NAC as indicated for 7 d were subjected to SA-β-gal staining (H) and EdU incorporation assay (I). (J) U2OS cells treated as in (H, I) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (K, L) U2OS cells treated as in (H, I) but 5 mM THFA instead of NAC were subjected to SA-β-gal staining (K) and EdU incorporation assay (L). (M) U2OS cells treated as in (K, L) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). (N, O) Hs68 cells treated as in (K, L) were subjected to SA-β-gal staining (N) and EdU incorporation assay (O). (P) Hs68 cells treated as in (N, O) but for 2 d instead of 7 d were subjected to immunoblot analysis. The protein levels were quantified as in (G). Data are mean ± SD (n = 3 independent experiments). Statistical significance is shown using the t test analysis; *P < 0.05, **P < 0.01, and n.s. = not significant (P > 0.05).
Tables
- Table 1.
Concentrations of free d-/l-amino acids in HepG2 cells treated with 10 μM etoposide for 48 h.
Form Concentration (μM) Ratio (Etoposide/DMSO) DMSO Etoposide Ala d N/D N/D — l 7791 10152 1.30 Arg d 100 130 1.3 l 120 160 1.33 Asp d Trace Trace — l 624 1006 1.61 Phe d N/D N/D — l 328 437 1.33 Pro d N/D N/D — l 2033 2439 1.20 Ser d Trace Trace — l 2312 3068 1.33 Tyr d N/D N/D — l 380 570 1.5 N/D, not detected; trace, detected but below limit of quantification.
