Research Paper Volume 14, Issue 21 pp 8839—8855

Figure 2. lncRNA HHIP-AS1 up-regulates HHIP expression and inhibits osteogenic differentiation of BM-MSCs. (A) The complementary sequence of the first exon of HHIP-AS1 and the first exon of HHIP. (B) lncRNA HHIP-AS1 in si-HHIP-AS1 or HHIP-AS1 transfected BM-MSCs was detected by PCR. (C) Fluorescence images of lncRNA HHIP-AS1 (Red), HHIP protein (Green) and DAPI (Blue) in BM-MSCs transfected with NC, si-HHIP-AS1 and HHIP-AS1. (D) HHIP mRNA in si-HHIP-AS1 or HHIP-AS1 transfected BM-MSCs detected by PCR. (E) HHIP protein in cell or in cell fractions of cytoplasm and cell membrane was detected by western blot after overexpression or knockdown of lncRNA HHIP-AS1. (F) RUNX2 and OCN as well as (G) ALP and OPN protein levels in cells were detected by western blot after overexpression or knockdown of lncRNA HHIP-AS1. (H) ALP and (I) alizarin red staining after 14 days of osteogenic induction of BM-MSCs. The histogram data for each group are the average of three independent replicates; bars represent standard deviation; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar = 10 μm. Note: Protein levels in lncRNA HHIP-AS1 knockdown and overexpression groups on day 7 were compared to those in NC groups on day7. Similarly, protein levels in lncRNA HHIP-AS1 knockdown and overexpression groups on day 14 were compared to those in NC groups on day14.