Fig 2: Chk1 inhibitor (GDC-0575) reduces CIP2A expression, activates PP2A, decreases tau phosphorylation and Aß levels in AD cell models. a Cell viability was detected in HEK293/tau cells by LDH cytotoxicity assay kit. b Representative immunoblots of Chk1, CIP2A, Tau-S199, Tau-S396, Tau-AT8, Tau-5, and ß-actin in HEK293/tau cells treated with increasing doses of GDC-0575 (0, 0.2, 0.5, 1 µM) for 24 h. Blots were from different gels with the same batch of samples electrophoresed. c The quantitative analysis of the protein levels in b. Non-phosphorylated proteins such as Chk1, CIP2A, and APP were normalized to the ß-actin levels; phosphorylated Tau-S199, -S396, and -AT8 were normalized to total tau (Tau-5) levels. d PP2A activity was detected in HEK293/tau cells. e Cell viability was detected in N2a/APP cells by LDH cytotoxicity assay kit. f Representative immunoblots of Chk1, CIP2A, APP, and ß-actin in N2a/APP cells treated with increasing doses of GDC-0575 (0, 0.2, 0.5, 1 µM) for 24 h. g The quantitative analysis of the protein levels in f. h PP2A activity was detected in N2a/APP cells. i The relative concentration of Aß40 in cell lysate and supernatant (left) and Aß42 in cell lysate and supernatant (right) of N2a/APP cells treated with 0.5 µM GDC-0575 treatment for 24 h. All data represent mean ± SEM, a–h n = 3, i the relative concentration of Aß40 and Aß42 in the cell lysate of the control group, n = 6, n = 4 respectively, and in the GDC group, n = 5, n = 4 respectively. i The relative concentration of Aß40 and Aß42 in supernatant of control group and GDC-0575 group n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to controls

Fig 3: Chk1 inhibitor (GDC-0575) ameliorates CIP2A upregulation, tau hyperphosphorylation and Aß production in the hippocampus of APP/PS1 mice. a Chk1 activity was detected in the hippocampus of the mice. b Representative immunoblots of ?H2A.X, 53BP1, CIP2A, Tau-AT8, Tau-T231, Tau-S262, Tau-5, APP, and ß-actin in the hippocampus of the mice. Blots were from different gels with the same batch of samples electrophoresed. c The quantitative analysis of the protein level in b. Non-phosphorylated proteins such as ?H2A.X, 53BP1, CIP2A, and APP were normalized to the ß-actin levels; phosphorylated Tau-S262, Tau-AT8, and Tau-T231 were normalized to total tau (Tau-5) levels. d PP2A activity was detected in the hippocampus of the mice. e The soluble Aß40 and Aß42 in the mice hippocampal tissues were detected by ELISA kit. f The insoluble Aß40 and Aß42 in the mice hippocampal tissues were detected by ELISA kit. All data represent mean ± SEM, a APP/PS1 group n = 4, b–f n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, comparison between the two groups. g Summary of the role of Chk1 activation in promoting AD-like pathologies through CIP2A/PP2A signaling

Fig 4: DNA damage, Chk1 activation, and increased CIP2A expression in AD human brains and AD mouse/cell models. a Representative immunoblots of ?H2A.X, 53BP1, Chk1-S345, Chk1-S296, Chk1-S317, Chk1, CIP2A, and ß-actin in AD brains. Blots were from different gels in which the same batch of samples was electrophoresed. b The quantitative analysis of the protein levels in a. Non-phosphorylated proteins such as ?H2A.X, 53BP1, total Chk1, and CIP2A were normalized to the ß-actin levels; phosphorylated Chk1-S345, -S296, and -S317 were normalized to total Chk1 levels. c Representative immunoblots of ?H2A.X, 53BP1, Chk1-S345, Chk1-S296, Chk1-S317, Chk1, CIP2A, and ß-actin in APP/PS1 mice brains. d The quantitative analysis of the protein levels in c. e Representative immunoblots of ?H2A.X, 53BP1, Chk1-S345, Chk1-S296, Chk1-S317, Chk1, CIP2A, and ß-actin in primary neurons with 2 µM Aß treatment for 48 h. f The quantitative analysis of the protein levels in e. g–h Correlation analysis of Chk1-S345 and ?H2A.X, Chk1-S345 and CIP2A in e. i The relative mRNA level of CIP2A in primary neurons with 2 µM Aß treatment for 12 h. All data represent mean ± SEM, n = 5 in (a–b); n = 3 in (c–i) *P < 0.05, **P < 0.01, ***P < 0.001, compared to controls. Statistical analyses details of all data are listed in the supplementary excel sheet

Fig 5: Overexpression of Chk1 in neurons induces CIP2A upregulation, tau/APP hyperphosphorylation, and synaptic impairments in mice. a Representative immunoblots of Chk1, CIP2A, Tau-S396, Tau-S404, Tau-5, APP, APP-T668, and β-actin in the hippocampus of the mice. Blots were from different gels with the same batch of samples electrophoresed. b The quantitative analysis of the protein level in a. Non-phosphorylated proteins such as Chk1, CIP2A, and APP were normalized to the β-actin levels; phosphorylated Tau-S396, Tau-S404, and APP-T668 were normalized to corresponding total tau (Tau-5) and APP levels respectively. c Representative images of Nissl staining of the mice brain slices. d The quantitative analysis of the neuron number in the CA1 region of c. The cell number was counted in brain slices from three mice in each group; one brain slice was counted for each mouse, and the neuron number in the CA1 region was counted by using the ImageJ software. e Representative picture in Golgi staining of the mice. f The quantitative analysis of the spine number in e from 3 brains. Spines in 9–10 intact dendrites in the hippocampal DG area were counted in each group. g Representative immunoblots of synaptic proteins (SYP, Synapsin I, GluA1 and PSD95) and GAPDH in the hippocampus of the mice. Blots were from different gels with the same batch of samples electrophoresed. h The quantitative analysis of the protein level in g. Bands intensity was normalized to GAPDH. All data represent mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared to controls