Fig 2: ARD1 restrains mTOR activity to promote autophagy through regulation of IKKβ.a MCF-7 and MDA-MB-231 cells were cotransfected with HA-ARD1, Ctrl siRNA, or IKKβ siRNA for 48 h, serum-starved overnight, and then treated with TNFα for 1 h. Cell lysates were collected for detection of LC3-I and LC3-II expression. β-Actin was used as a protein loading control. b Cells were transfected with HA-Ctrl, HA-ARD1 WT, or HA-ARD1 mutants, serum-starved overnight, and then treated with TNFα for 1 h. Treated cells were determined as described in a. c MCF-7 cells were cotransfected with GFP-LC3 and HA-Ctrl, HA-ARD1 WT, or HA-ARD1 S209A, serum-starved overnight, and then treated with TNFα for 1 h. Treated cells were fixed, and then visualized by fluorescent microscopy. Bars, 10 µM. d MCF-7 and MDA-MB-231 cells were cotransfected with HA-ARD1 and myc-Rheb. Cell lysates were collected for LC3-I and LC3-II expression detection. e Cells were transfected with HA-ARD1 and then treated with the inhibitor of class III PI3 kinases 3-MA (5 mM) for 4 h, lysed and subjected to western blotting with anti-LC3 antibodies to monitor autophagy. f and g Cells were treated as described in d and e, respectively. Cell viability and proliferation were determined. Graphs showing the results of quantitative analyses (n = 3, mean ± S.D. *P < 0.05)

Fig 3: Activity measurement of NatA from wildtype (WT) and Naa10 knockout (KO) mice.(A) Correlation of Naa10 alteration state on amino-terminal acetylation in mouse embryonic fibroblasts (MEFs). Each dot (n = 533) represents the average amino-terminal acetylation percentage of five replicates of Naa10+/Y and Naa10-/Y, respectively. Dashed lines are the borders of ±10% difference. Except for the 10 dots, 522 of the 533 dots are within the borders. The marginal histograms show the distribution of amino-terminal acetylation data points. (B) Immunoprecipitation of Naa15. Liver tissue from WT and Naa10 KO mouse was lysed and incubated with anti-Naa15 antibody to retrieve NatA complexes. Proteins were separated by SDS-PAGE and immunoblots probed with anti-Naa15 antibody and anti-NAA10 antibody. (C) Catalytic activity of immunoprecipitated NatA. The catalytic activity of NatA precipitated from WT and Naa10 KO mouse liver tissue by anti-Naa15 was measured towards the NatA substrate peptide SESS24 in an in vitro [14C]-Ac-CoA–based acetylation assay. Control reactions were performed with no enzyme or no peptide to account for background signal. The immunoprecipitation (IP) and activity measurements were performed in three independent setups, each with three technical replicates per assay. One representative setup is shown. Figure 3—source data 1.Identification of a potential Naa10 homolog.

Fig 4: ARD1 inhibits the mTOR activity through IKKβ regulation.a Cells were transfected with Ctrl siRNA, IKKβ, or IKKα siRNA for 48 h, serum-starved overnight, and then treated with TNFα for 1 h. Treated cells were collected for western blot analysis of the expression of the protein shown in the figure. β-Actin was used as a protein loading control. p-S6K1T389 means the phosphorylation status of S6K1. b Cells were transfected with HA-Ctrl, HA-ARD1 WT, or HA-ARD1 mutants for 48 h, serum-starved overnight, and treated with TNFα for 1 h. Treated cells were collected for western blot analysis of the expression of the protein shown in the figure. c Cells were cotransfected with FLAG-IKKβ, HA-Ctrl, HA-ARD1 WT, or HA-ARD1 mutants for 48 h, serum-starved overnight, and treated with TNFα for 1 h. Treated cells were collected for the detection of protein expression. d Lysates of HEK293T cells cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-S6K1 were analyzed with antibodies directed against the HA tag by coimmunoprecipitation and immunoblotting by p-S6K1T389 and HA. e HEK293T cells were cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-4EBP1, and then the cells were lysed for coimmunoprecipitation assay as described in d. f HEK293T cells were cotransfected with FLAG-IKKβ, Myc-ARD1, and HA-TSC2 and then treated as described in d. g MCF-7 cells were serum-starved overnight and treated with TNFα for 1 h. Cell lysates were immunoprecipitated with specific antibodies to ARD1 and TSC2 to identify the association of endogenous proteins. h Indicated cells were transfected with HA-ARD1 WT or HA-ARD1 S209A, serum-starved overnight, and then treated with TNFα for 1 h. Cell lysates were collected for the detection of the change of p-S6K1T389. i Treated cells as described in h were collected for the detection of cell viability and proliferation. Graphs showing results of quantitative analyses (n = 3, mean ± S.D. *P < 0.05)

Fig 5: Pleiotropic phenotypes of Naa10 knockout (KO) mice.(A–C) Representative images of abnormalities in Naa10-/Y compared with Naa10+/Y. (A) Body weight of male (left) and female (right) versus ages was monitored from 2 weeks. The weight of Naa10-/Y and Naa10-/- mice is markedly reduced compared with that of the wildtype (WT) mice. Asterisks indicate a statistical difference calculated by Student’s t-test: *p<0.05. (B) Representative images of completely penetrant phenotypes. Hypopigmentation (Naa10+/Y, n = 243; Naa10-/Y, n = 121; Naa10tm1a/Y, n = 17) and supernumerary ribs (Naa10+/Y, n = 3; Naa10-/Y, n = 6; Naa10tm1a/Y, n = 2; E18.5) were found 100% in Naa10-deficient mice. (C) Naa10-/Y is smaller in size and has round-shaped head (Naa10+/Y 0/59, Naa10-/Y 7/33). Over time, hydrocephaly became apparent (N = 14/29 [~48%] for >P7 male Naa10-Y; N = 7/19 [~36%] for >P7 female Naa10-/-). Hydronephrosis (red arrow, Naa10+/Y 0/23, Naa10-/Y 14/29, Naa10+/+ 0/5, Naa10-/- 7/19) and abnormal genitalia (black arrow) of male (middle, Naa10+/Y 0/23, Naa10-/Y 16/29) and female (bottom, hydrometrocolpos, Naa10+/+ 0/5, Naa10-/- 7/19) are shown.