Fig 2: HPC reduces the expression of DNMT3A, inhibits the DNA methylation of CREB2 and increases the expression of CREB2 in CA1. (A) Western blot analysis of DNMT3A expression in CA1. (B) Representative images of Western blot show the expression of DNMT3A in CA1 after tGCI with and without Piwil2 AS‐ODN administration. (C) Representative immunoblots of DNMT3A expression in CA1 after HPC with and without Piwil2 lentiviral vectors administration. (D) Representative immunoblots of CREB2 expression in CA1. (E) Representative images of Western blot show the expression of CREB2 in CA1 after tGCI with and without Piwil2 AS‐ODN administration. (F) Representative immunoblots of CREB2 expression in CA1 after HPC with and without Piwil2 lentiviral vectors administration. (G) RT‐qPCR analysis of CREB2 mRNA in CA1. (H) RT‐qPCR analysis of CREB2 mRNA in CA1 after tGCI with and without Piwil2 AS‐ODN administration. Data are expressed as percentage of value of Sham animals. Each bar represents the mean ± SD. *p < 0.05 vs. Sham animals (A–H), # p < 0.05 vs. tGCI groups at the same time point (A,D,G). # P < 0.05 vs. tGCI group and & p < 0.05 vs. tGCI group or HPC group administrated with aCSF (B,E,H), and & p < 0.05 vs. Sham or HPC group with injection of Lenti‐Con (C,F). (I) Methylation status of CpG sites in the CREB2 promoter region (from +1540 bp to +1943 bp) determined using MSP and BSP analysis. (J) The methylation status of CREB2 gene was analyzed by MSP using two different sets of primers. U, unmethylated PCR products; M, methylated PCR products. PCR products are about 200 bp. (K) Quantification of the frequency of methylation at each CpG site of CREB2 promoter. Data are expressed as percentage of total CpG methylation sites between +1540 bp to +1943 bp of the CREB2 promoter. Each bar represents the mean ± SD. *p < 0.05 vs. Sham animals, # p < 0.05 vs. tGCI group and & p < 0.05 vs. tGCI group or HPC group. Black dot, methylated; white dot, unmethylated. AS‐ODN, antisense oligodeoxynucleotide; CREB2, cyclic AMP response element‐binding 2; DNMT3A, DNA methyltransferase 3A; HPC, hypoxic postconditioning; Lenti‐Con, Lenti‐Control: scrambled lentivirus vector; lenti‐Piwil2, Piwil2‐carried lentivirus; Sham, sham‐operated; tGCI, transient global cerebral ischemia

Fig 3: HPC enhances the plasticity of hippocampal dendritic spines and improves memory function after tGCI. (A) Representative reconstructions of CA1 pyramidal neurons using Golgi staining. Magnified views of the red‐boxed regions on middle column are z‐stack images of dendrites. Magnified views on the right show structural changes in dendritic spines during tGCI or HPC with and without Piwil2 AS‐ODN administration. Asterisk indicates a spine partially collapsed down into the dendritic shaft during tGCI. (B,C) Sholl analyses of dendrites in CA1 pyramidal neurons. (D,E) Quantification of neuronal morphometric analyses, length of dendritic branch and density of dendrite spines. Cognitive function was tested by Morris water maze. (F) Representative path tracings in each quadrant during the training period (learning) and the probe trial (memory). The swimming speed (G), path length (H) and escape latency (I) were recorded during the training period. (J) The percentage of time spent in the target quadrant to total time (30 s) was recorded at day 7 after tGCI. Data are expressed as the mean ± SD. *p < 0.05 vs. sham animals, # p < 0.05 vs. tGCI group, and & p < 0.05 vs. tGCI or HPC group. aCSF, artificial cerebrospinal fluid; AS‐ODN, antisense oligodeoxynucleotide; HPC, hypoxic postconditioning; Sham, sham‐operated; tGCI, transient global cerebral ischemia

Fig 4: Piwil2 initiates cell reprogramming by regulating the balance between the acetylation and trimethylation of H3K9a. Western blot showing significantly induced H3K9 acetylation but reduced H3K9 trimethylation in HaCaT cells transfected with Piwil2 or E6 and E7 compared with those only transfected with vector. b. Co-immunoprecipitation showing that Piwil2, either overexpressed or induced by E6 and E7, interacted with acetylated H3K9. c. EMT markers upregulated in SiHa cells exhibiting Piwil2 overexpression but downregulated in those cells in which Piwil2 was knocked down, as verified by qRT-PCR. d. The proportion of ALDH-, MSCA-1-, and ABCG2-positive cells, determined by FACS in cells with Piwil2 overexpression or knockdown. e. The LD50 dose of cisplatin, detected by CCK8 assay in cells with Piwil2 overexpression or knockdown. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Fig 5: Piwil2 overexpression induces HaCaT cell malignant transformationa. HaCaT cells were transfected with lentivirus containing Piwil2 or lentiviral vector, and cell numbers were plotted daily. b.-c. Colony formation numbers and invading HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. d. Proteins were analyzed by western blotting for c-Myc, E-cadherin, and molecules regulating cell proliferation and apoptosis. e.-f. The expression of EMT markers was determined by qRT-PCR and western blotting in HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.