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. 2017 Aug 28;1(8):816–830. doi: 10.1002/hep4.1086

Figure 3.

Figure 3

miR‐127 represses HMGB2 protein expression to facilitate EB body formation. (A) Sequence alignment of miR‐127 and the 3'UTR of HMGB2 in human, mouse, and rat. The seed‐match region is underlined. (B) Transient transfection assays. Left, HeLa cells were transfected with HMGB2 3'UTR luciferase reporter along with miR‐127 expression plasmid. Middle, HeLa cells were transfected with WT or mutant HMGB2 3'UTR with or without miR‐127. Right, HeLa cells were transfected with HMGB2 3'UTR with or without miR‐127 antagomir. Luciferase reporter activity was normalized to Renilla activity. (C) Western blot to determine endogenous HMGB2 protein in HeLa cells that were transfected with pTarget, pTarget‐miR‐433, or pTarget‐miR‐127. (D) Western blot to determine HMGB2 and CREB proteins (left) and qPCR to determine miR‐127 expression (right) in MHCC97H cells transfected with (+) or without (–) miR‐127. (E) Western blot to determine HMGB2 and CREB proteins (left) and qPCR to determine miR‐127 expression (right) in HepG2 cells transfected with or without miR‐127. (F) Western blot to determine HMGB2 protein (left) and qPCR to determine miR‐127 expression (right) in HepG2 cells transfected with or without miR‐127 antagomir (anti‐miR‐127). (G) Left, qPCR of mRNA expression of pluripotency markers and mesoderm markers in undifferentiated CGR8 cells and EB‐4D after transduction with GFP control or GFP‐miR‐127 lentivirus. Data are shown as mean ± SEM (triplicate assays). *P < 0.05 versus corresponding control. Right, western blot of protein expression. Abbreviations: CREB, cyclic adenosine monophosphate response element‐binding protein; mut, mutant; hr, hours; qPCR, quantitative polymerase chain reaction.