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. 2018 Jul 6;9:1557. doi: 10.3389/fimmu.2018.01557

Figure 7.

Figure 7

CASC2c regulated factor X (FX) expression and macrophage migration and polarization. (A) Real-time PCR detected decreased miR-338-3p expression in G1124 cells when transfected with CASC2c (mean ± SEM, *p < 0.05). (B) Real-time PCR detected reduced FX expression in G1124 cells with overexpression of CASC2c (mean ± SEM, **p < 0.01). (C) Western blotting detected reduced FX expression in G1124 cells with overexpression of CASC2c. (D) Real-time PCR detected decreased CASC2c expression after transfection with miR338-3p mimics (mean ± SEM, *p < 0.05). (E) The luciferase reporter assay showed that the effect of miR-338-3p on CASC2c activity (mean ± SEM, **p < 0.01). (F) Overexpression of CASC2c in G1124 cells suppressed the migration of THP-1 cells toward G1124 cell supernatants (mean ± SEM, **p < 0.01). (G) Real-time PCR detected the M1 markers and M2 markers in THP-1 cells after incubation with G1124 cell supernatants that were transfected with CASC2c or control vector. M2 markers (MRC1, SerpinB2, and STAB1) were decreased, while M1 markers (IL-1β, IL-12α, CXCL9, and iNOS) in THP-1 cells were increased when CASC2c was overexpressed in G1124 cells (mean ± SEM, *p < 0.05, **p < 0.01). (H) Knockdown of CASC2c increased FX mRNA expression in U251 cells (mean ± SEM, *p < 0.05). (I) Knockdown of CASC2c increased FX protein detected by Western blotting (mean ± SEM, *p < 0.05). (J) Knockdown of CASC2c recruited more THP-1 cells measured by transwell assay (mean ± SEM, *p < 0.05). (K) M1 markers and M2 markers in THP-1 cells were measured by real-time PCR after incubated with U251 cell supernatants transfected with sh-CASC2c or sh-NC vector. The mRNA expression of M1 markers (IL-12α, CXCL9, and CXCL10) were decreased, while M2 markers (HMOX1, MRC1, and STAB1) were increased when THP-1 cells were incubated with U251 cell supernatants transfected with sh-CASC2c (mean ± SEM, *p < 0.05, **p < 0.01). (L,M) Flow cytometry revealed that the proportion of CD11b+CD80+ M1 macrophages increased and CD11b+CD206+ M2 macrophages decreased when THP-1 treated with supernatants from G1124 cells transfected with CASC2c compared with control vector. (M) Bar graph of CD11b+CD80+ and CD11b+CD206+ cell proportion in panel (L).