Figure.7

MEK6-AS1 potentially modulates mRNA stability of MEK6. a qRT-PCR and western blot were used to verify MEK6 expression in cells with MEK6-AS1 knockdown and control group during adipogenic differentiation. It should be emphasized that the GAPDH band is same to Fig. 3C. b qRT-PCR and western blot were used to verify MEK6 expression of cells infected with MEK6-AS1 overexpression virus or control virus during adipogenic differentiation. It should be emphasized that the GAPDH band is same to Fig. 3I. c and d qRT-PCR analyzed expression of MEK6-AS1 in hAMSCs after knockdown (c) or overexpression (d) of MEK6. e and f qRT-PCR (e) and western blotting (f) detected adipogenic markers in cells after MEK6 knockdown followed by MEK6-AS1 overexpression. g Western blotting detected adipogenic markers in cells with Adezmapimod (a MAPK pathway inhibitor) followed by MEK6-AS1 overexpression. h and i Silver staining showed that there was one differential band each between sense and anti-sense of MEK6-AS1 (h) and MEK6 mRNA (i). The red arrows indicated differential bands.(j and k) qRT-PCR of MEK6 stability in the presence of MEK6-AS1 knockdown (j) and overexpression (k) in cells treated with Actinomycin D for 0 h, 2 h, 4 h, 6 h and 8 h. l and m qRT-PCR of MEK6-AS1 stability (l) and MEK6 stability m in NAT10 silenced cells and control cells treated with Actinomycin D for 0 h, 2 h, 4 h, 6 h and 8 h. n qRT-PCR detected adipogenic marker genes in cells after NAT10 knockdown followed by MEK6-AS1 overexpression. o Schematic representation of the mechanism that MEK6-AS1 promotes adipogenic differentiation of hAMSCs by regulating MEK6 mRNA stability. GAPDH was used as internal control for qRT-PCR and western blot. The quantitative data were normalized to GAPDH. n = 3. Data are shown as the mean ± SD. Statistically significant differences were considered as follows: ∗P < 0.05, ∗  ∗P < 0.01, ∗  ∗  ∗P < 0.001, ∗  ∗  ∗  ∗P < 0.0001, ns: not significant