The effect of Cldn-1 knockdown on osteoblast differentiation in MC3T3-E1 cells.

A) ALP activity was determined in MC3T3-E1 cells transduced with control or Cldn-1 shRNA and treated with βGP ± AA for 72 hrs. Values (means ± SEM; n = 8). A = <0.05 vs. βGP treated, and B = <0.05 vs. control shRNA at the corresponding treatment. B) ALP staining was determined on MC3T3-E1 cells transduced with control or Cldn-1 shRNA and treated with βGP ± AA for 6 days, followed by ALP activity staining. Values (means ± SEM; n = 5) are represented as % ALP stained area. A = <0.05 vs. βGP treated, and B = <0.05 vs. control shRNA at the corresponding treatment. C) The expression of osteogenic master transcription factor genes (osterix and Runx-2) and osteogenic marker genes (ALP, bone sialoprotein (BSP), and osteocalcin) was evaluated in control and Cldn-1 shRNA MC3T3-E1 treated β-glycerophosphate (βGP) with or without ascorbic acid (AA) for 24 hrs and 6 days using real time RT-PCR. Values (means ± SEM; n = 4) are presented as % of control shRNA. A = <0.05 vs. control shRNA cells.