Fig 2: Spatiotemporal expression of ANXA3 protein and mRNA. (a, b) The expression levels of ANXA3 protein in rat embryos from E9.5 to E19.5 were determined by Western blotting analysis. The panel shows the protein bands corresponding to ANXA3 and tubulin. The histogram indicates the quantification of the densitometric analysis. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. ∗p < 0.05 and ∗∗p < 0.01 compared with the E9.5 group. n = 6 per group. (c) Real-time PCR analysis of ANXA3 mRNA expression in rat embryos from E9.5 to E19.5. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. ∗p < 0.05 and ∗∗p < 0.01 compared with the E9.5 group. n = 6 per group. (d–i) The levels of ANXA3 mRNA expression in the olfactory bulb, cortex, hippocampus, thalamus, cerebellum, and spinal cord of rats aged 1, 3, 6, 9, and 12 months were determined by qPCR analysis. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the 1 m group. n = 6 per group. 1 m: 1 month; 3 m: 3 months; 6 m: 6 months; 9 m: 9 months; 12 m: 12 months.

Fig 3: Morphologies of microglia labeled by ANXA3 and ANXA3 protein expression levels in different regions. (a) Low-magnification micrographs showing microglial cells in the ischemic penumbra 3 days after reperfusion. The yellow-dotted ring indicates the ischemic penumbra area between the infarct area and the normal area of the brain tissue. Scale bars = 200 μm. (b) Representative immunofluorescence images showing the morphology of activated microglia in the ischemic penumbra (on the left) and resting microglia in the healthy area (on the right) labeled by ANXA3 and Iba1 at high magnification. Scale bars = 50 μm. n = 5 per group. (c, d) The expression levels of ANXA3, CD11b, and Iba1 proteins in the olfactory bulb, cortex, hippocampus, thalamus, cerebellum, and spinal cord of adult rats (3 months) were determined by Western blotting analysis. The panel shows protein bands corresponding to ANXA3, CD11b, Iba1, and tubulin. The histogram shows the results of the densitometric analysis. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the cerebellum group. ##p < 0.01 and ###p < 0.001 compared with the cerebellum group. &p < 0.05, &&p < 0.01, and &&&p < 0.001 compared with the cerebellum group. n = 6 per group.

Fig 4: Subcellular localization of ANXA3 in resting and activated N9 microglial cells and lentiviral transfection of N9 cells. (a) Representative immunofluorescence images showing ANXA3 expression in microglial cells. N9 cells were also stained for CD11b expression. The inset shows the microglia at a higher magnification. Scale bar = 40 µm. (b, c) Western blotting analysis of ANXA3 protein expression levels in the cytoplasm and nuclei of resting (Con) and LPS-induced activated microglia (500 ng/ml LPS treatment for 24 hours). The panel shows the ANXA3, tubulin, and histone H3 protein bands, and the histogram reflects the results of the densitometric analysis. The data are expressed as the means ± SD and were analyzed by Student's t-tests. ***p < 0.001 compared with the control group. The data were pooled from six independent experiments.

Fig 5: The proliferation of microglial N9 cells. (a, b) Flow cytometry analysis showing the proliferation of the Con, LV-empty, LV-NC, LV-ANXA3, and LV-shANXA3-treated N9 microglial cells. MultiCycle AV software was used to perform the fit analysis of the DNA cell cycle and to calculate the percentages of cells in each phase. The blue peak, the green arc-shaped hatched portion in the middle, and the red peak represent the G1, S, and G2 phases of the cell cycle, respectively. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. **p < 0.01 and ***p < 0.001 compared with the LV-empty group. ##p < 0.01 and ###p < 0.001 compared with the LV-NC group. The data were pooled from five independent experiments. (c, d) The EdU incorporation assay showing the proliferation of the Con, LV-empty, LV-NC, LV-ANXA3, and LV-shANXA3-treated N9 microglial cells. The cell nuclei were stained with DAPI. EdU-positive cells are indicated in pink. The data are expressed as the means ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. **p < 0.01 compared with the LV-empty group. ##p < 0.01 compared with the LV-NC group. The data were pooled from five independent experiments. Scale bar = 20 µm. DAPI: 4',6-diamidino-2-phenylindole; EdU: 5-ethynyl-2'-deoxyuridine.