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. 2018 Jan 29;9(2):66. doi: 10.3390/genes9020066

Figure 2.

Figure 2

WNT9A increases hematopoietic differentiation efficiency in a time-dependent manner. (A) Schematic of inducible WNT9A transgene, which consists of a doxycycline-inducible WNT9A and an internal ribosome entry site (IRES) driving expression of Citrine. (B) Immunoblot depicting WNT9A protein expression dynamics in response to 50 ng/mL doxycycline treatment. Cells were treated with doxycycline for 0, 12, 24, or 48 h and analyzed for expression of WNT9A. The 72-h sample was treated with doxycycline for 48 h, then changed to fresh media without doxycycline for 24 h. Top: Conditioned media was pulled down with Blue Sepharose beads and blotted for WNT9A; Middle: Cell lysate was blotted for WNT9A; Bottom: Cell lysate was blotted for β-ACTIN. (C) Schematic of doxycycline treatment (Tx) to induce WNT9A expression, and readout of hematopoietic progenitor differentiation by flow cytometry for CD34 and CD45 for the data shown in (D,E). Red arrows indicate doxycycline treatment, black diamonds indicate the resultant WNT9A protein expression. (D) Representative flow cytometry plots for data graphed in (E); (E) Percentage of CD34+/CD45+ cells normalized to untreated control. Data represent average of three independent biological replicates; ** = p value < 0.01, n.s. = Not significant. Error bars represent standard deviation.