Fig 2: Restoration of SATB2 expression abolishes the antitumor effects of miR-3666. (A) Protein expression of SATB2 was detected by western blotting in SW480 and HCT116 cells cotransfected with miR-3666 mimics, miR-3666 mimics+pcDNA3.1-SATB2 or negative controls. (B) Cell proliferation was detected via a CCK-8 assay. (C and D) Cell migration and invasion were detected via Transwell assays (magnification, ×100). *P<0.05. CCK-8, Cell Counting Kit-8; miR, microRNA; NC, negative control; oe, overexpression; SATB2, special AT-rich sequence binding protein 2.

Fig 3: SATB2 is a target gene of miR-3666. (A) A diagram for the potential binding site of miR-3666 in the 3′-UTR of SATB2 mRNA. (B) A SATB2 3′-UTR fragment containing the Wt or Mut miR-3666 binding site was cloned into the pmirGLO reporter gene vector. Cells were cotransfected with the reporter gene construct vector and miR-3666 mimics or inhibitor for 48 h. (C and D) HCT116 and SW480 cells were transfected with miR-3666 mimics or inhibitor for 48 h. Relative mRNA and protein levels of SATB2 were detected by RT-qPCR and western blotting. (E) Correlation between SATB2 mRNA and miR-3666 expression was assessed by Pearson's correlation analysis. (F) Relative expression of SATB2 was measured by RT-qPCR in CRC tissues and adjacent normal tissues. (G) SATB2 expression was determined by immunohistochemical analysis in paired CRC tissues and normal tissues (magnification, ×100). (H) Western blot analysis of SATB2 protein expression in 3 pairs of CRC tissues and adjacent normal tissues. *P<0.05 vs. NC group or normal tissues. miR, microRNA; Mut, mutant; N, normal tissue; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SATB2, special AT-rich sequence binding protein 2; T, tumor tissue; UTR, untranslated region; Wt, wild type.

Fig 4: Increased death of immature neurons in Area 46 of ZIKV-infected animals.Representative tissue samples from Area 17 (A–L) and Area 46 (M–Z) in control and ZIKV-infected animals. Immature neuronal marker SATB2 (A, G, M, S) colocalization with CC3 (B, H, N, T), an apoptotic marker, and DAPI (C, I, O, U) were analyzed in Areas 17 and 46 of control and ZIKV-infected animals. Merged images (D, J, P, V). 3D surface rendering reconstruction of SATB2 and CC3 are shown in E, K, Q, and X. Average total cell counting (F, R) and quantification of SATB2/CC3 colocalization (L, Z) indicated no group differences in the number of immature neurons undergoing apoptosis in Area 17 (L), but a significantly greater immature neurons undergoing apoptosis in ZIKV-infected compared to control animals in Area 46 (R). Scale bar = 25 µm.