Figure 7

Effectiveness of Pax6 knockdown and overexpression. (A) Schematics show small hairpin RNA (shRNA) target location in Pax6 and Pax6 5a and the structure of the U6 shRNA construct (bottom). (B) Western blots show Pax6 shRNA knockdown in transfected 293T cells. DNAs used are listed on the top and antibodies used are on the left of the blots. U6, control vector used to express shRNA; Pax6i, U6 vector expressing shRNA targeting Pax6; Redi1 and Redi2, U6 vectors expressing shRNAs targeting red fluorescent protein (RFP). (C) Immunolabeling shows Pax6 protein knockdown in transfected E4.5 (HH stage 24) retinal cells after 2 days in vitro (DIV). Pax6i transfected samples showed fewer cells co-expressing green fluorescent protein (GFP) and Pax6. (D) Quantification of Pax6 protein signal intensities among U6 vector and U6-Pax6i vector transfected E4.5 retinal cells after 2 DIV. Asterisks represent P-values between the control and the Pax6i samples: *P < 0.05 (N ≥ 4, mean ± standard error). (E) Immunofluorescent staining of E5 (HH stage 27) retinal cells transfected with CAG control vector and Pax6 or Pax6(5a) overexpression vectors after 2 DIV. Note both Pax6 and Pax6(5a) expression vector-transfected cells show higher Pax6 expression levels than endogenous Pax6 levels observed in retinal cells.