Figure 4: Effect of BBS1 and BBS10 knockdown on cilium integrity, microtubule organization and cell polarity in MCD4 cells. MCD4 cells were either mock-transfected or transfected with negative control siRNA or specific anti BBS1 or BBS10 siRNA. (A) Silencing efficiency was tested 48 h after transfection by semiquantitative RT-PCR and the effect of BBS1 or BBS10 silencing evaluated by densitometric analysis of the RT-PCR bands. (B) Compared to control siRNAtransfected cells, BBS1 and BBS10 silencing dramatically reduced the number of ciliated cells as indicated by the staining of acetylated tubulin at the top of the cells (white arrowheads, apical). In a subapical confocal section, the staining of acetylated tubulin was dramatically reduced in BBS10-silenced cells. The intensity and localization of the tight junction marker Zo-1 and the basolateral marker Na+/K+-ATPase were virtually unaffected by BBS silencing. Experiments were repeated at least three times with comparable results.