Fig. 4

CircAPP regulates CLIC1 expression and channel activity through miR-1906. (A) RT-PCR assay was performed to detect circAPP expression in the cytoplasm and nucleus of BV-2 cells, n = 3. (B) The subcellular location of circAPP in BV-2 cells was detected using RNA-FISH assay. (C, D) The interaction between circAPP and miR-1906 was assessed using RNA pull-down assay, n = 4. (E) The effect of circAPP knockdown in microglia on miR-1906 expression in the hippocampus of APP/PS1 mice was evaluated using RT-PCR assay, n = 4. (F) The interaction between miR-1906 and 3’UTR of CLIC1 mRNA was assessed using RNA pull-down assay, n = 3. (G ~ I) The effects of miR-1906 overexpression on CLIC1 total expression in Aβ-treated BV-2 cells (G) and the hippocampus of APP/PS1 mice (H) and membrane expression in Aβ-treated BV-2 cells (I) were evaluated using Western blot assay, n = 4. (J) The effect of miR-1906 overexpression on CLIC1 channel activity in Aβ-treated BV-2 cells was assessed using functional chloride channel assay, n = 6. (K, L) The effects of miR-1906 overexpression on total (K) and membrane (L) expression of CLIC1 regulated by circAPP knockdown in Aβ-treated BV-2 cells were assessed using Western blot assay, n = 4. (M) The effect of miR-1906 overexpression on CLIC1 channel activity regulated by circAPP knockdown in Aβ-treated BV-2 cells was assessed using functional chloride channel assay, n = 6. All data in the figure are presented as mean ± SEM. Student’s t test (C ~ F, H) and one-way ANOVA (G, I ~ M) were used to assess statistically significant differences. *P < 0.05, **P < 0.01