Fig 2: Id2 competitively interrupts the association of Sirt2 with a-tubulin.A, B GST–Id2 and mammalian GFP–Sirt2 fragments (A) transfected cells were subjected to GST pull-down assay and immunoblotting as indicated. C, D Flag–Sirt2 and mammalian GST–a-tubulin fragments (C) transfected cells were subjected to GST pull-down and immunoblotting as indicated. E, F HEK293T cells were transfected with 0, 1, 3 µg of GFP–Id2 (E) or Flag–Sirt2 (F) with GST–a-tubulin, and lysates were subjected to GST pull-down and immunoblotting. The bar graphs represent the quantification of Sirt2 or Id2–a-tubulin binding. G, H Primary hippocampal neurons were transfected as indicated and the cell lysates were subjected to GST pull-down assay. I Proximity ligation assay (PLA) was conducted with scr, si-Id2, or si-Sirt2 expressing cells. Confocal images were shown tubulin-Id2 PLA staining (left) and tubulin-Sirt2 PLA staining (right). Nuclei were stained by DAPI (blue). Scale bar 5 µm. Quantification of tubulin-Id2 or tubulin-Sirt2 PLA puncta is shown as a bar graph. **p < 0.005, ***p < 0.0005, ****p < 0.0001. All values are mean ± SEM and the representative images shown are from at least three independent experiments.

Fig 3: Id2 protects aK40 acetylation by blocking Sirt2-mediated deacetylation.A HEK293T cells were transfected as indicated and the cell lysates were subjected to GST pull-down and immunoblotting as indicated. B In vitro deacetylase assay was performed with GFP–Id2 and Flag–Sirt2 proteins. The bar graphs represent the quantification of the acetyl-a-tubulin to a-tubulin ratio. C, D GST–Id2 and GFP–HDAC6 (C) or GFP–aTAT (D) transfected cell lysates were subjected to immunoblotting as indicated. E Primary cultured neurons were co-transfected with GFP–Sirt2 or GFP vector control and RFP–Id2 or RFP vector on DIV 3 and fixed on DIV 5. Neurons were stained with anti-NeuN (blue) and anti-acetylated tubulin (purple). Quantification of fluorescence intensity and axon length is shown at the right. Scale bar, 20 µm. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. All values are mean ± SEM and the representative images shown are from at least three independent experiments.

Fig 4: Effects of Sirt2 on PADI3-expressing HCT116 cells. PADI3-expressing HCT116 cells were transfected with pCDNA3.1-Sirt2-iso1 plasmids (OE-PADI3-Sirt2-iso1) or pCDNA3.1-Sirt2-iso2 plasmids (OE-PADI3-Sirt2-iso2) separately. PADI3-expressing HCT116 cells were used as the positive control, and RFP-expressing HCT116 cells were used as the negative control. (A) Western blot analysis was used to measure the expression of AKT, p-AKT, Snail and p21. (B) A CCK-8 assay was used to measure cell proliferation. (C) The cell cycle was analyzed using FCM. (D) Statistical analysis of C. (E) Colony number and size were analyzed. (F) Statistical analysis of E. * indicates P < 0.05 for three independent experiments analyzed by Student’s t-test.

Fig 5: mRNA expression of Beclin1 (A), Atg5 (B), and SIRT2 (C) in the cerebral cortex, hippocampus, and cerebellum of young and aged groups. The SIRT2 expression was increased in all three brain regions and beclin1 in the cortex of aged rats. For each group, n = 6, and the data were presented as mean ± SEM. *refers to significant difference at the level of p < 0.05 compared to the young control group, #refers to significant difference at the level of p < 0.05 compared to the aged control group.