Research Paper Volume 13, Issue 10 pp 13807—13821

Figure 6. Exploration of the up- and downstream signal pathways regulated by AOBEE. (A) Signal pathway inhibitors were used to detect the changes of lnc865 and lnc556 expression. SB431542 and SB203580 inhibited lnc865 and lnc556 expression. (B) Western blot showed that AOBEE decreased p38MAPK, smad2/3 and TGFβ1 receptor II expression in in vivo and in vitro models. (C) Identification of downstream signal pathways affected by AOBEE in L929 cells. The x- and y-axes represent the normalized ratio of firefly/Renilla luciferase activities, respectively. KLF4 is one of the significant changes of signal pathways. (D) AOBEE did not cause any changes of KLF4 expression at the mRNA level by using qRT-PCR. (E) Western blot showed that AOBEE repressed KLF4 expression at the protein level. (F) Si-lnc865 and si-lnc556 did not cause any changes of KLF4 expression at the mRNA levels by using qRT-PCR. (G) Western blot showed that si-lnc865 and si-lnc556 repressed KLF4 expression at the protein level. (H) The degradation of KLF4 was detected by using the protein synthesis inhibitor actidione in L929 cells. The fastest degradation rate was in the normal group and the slowest degradation rate was in the TGFβ1 group. AOBEE promoted KLF4 degradation compared with TGFβ1. (I) Immunofluorescence experiments verified that AOBEE, si-lnc865, and si-lnc556 inhibited KLF4 expression at the protein level. (J) MiR-29b-2-5p inhibitor increased KLF4 expression and miR-29b-2-5p mimic decreased KLF4 expression. (K) The rescue experiments showed that si-lnc865 and si-lnc556 decreased KLF4 expression, and miR-29b-2-5p inhibitor increased KLF4 expression at the protein level. Each bar represents the mean ± SD, n = 6; *p < 0.05, **p < 0.01.