RABEP2-PACT does not rescue siSDCCAG8 ciliogenesis defect.

(A,B) hTERT-RPE1 cells were transfected with non-specific siRNA (siNS) and 24 hours later transfected with EGFP-PACT (A) or EGFP-RABEP2-PACT (B) constructs. Either of the tagged proteins localized to the centrosome and were detected with anti-GFP antibody (green). Ciliogenesis (acetylated-α-tubulin, red) was not affected in either of the situations. (C,D) hTERT-RPE1 cells were transfected with siSDCCAG8 and 24 hours later transfected with EGFP-PACT (C) or EGFP-RABEP2-PACT (D) constructs. Although, the tagged proteins localized to the centrosome (green), they failed to rescue the ciliogenesis defect (acetylated α-tubulin, red) in SDCCAG8 knockdown cells. Scale bars: 4 μm. (E) Quantitation of the percentage of ciliated cells in (A, B, C and D), demonstrates that over expression of the EGFP-PACT or EGFP-RABEP2-PACT construct does not affect normal ciliation in siNS hTERT-RPE1 cells. Moreover, EGFP-RABEP2-PACT construct fails to rescue the ciliation defect in SDCCAG8 depleted cells (**p = 0.0068). Error bars, SEM. (F) We measured the length of cilia in EGFP-PACT and EGFP-RABEP2-PACT overexpression cells to examine whether it was affected. There was no significant difference in the length of cilia between siNS cells expressing EGFP-PACT and EGFP-RABEP2-PACT (73.08 ± 4.417 μm, n = 3, vs. 67.80 ± 2.446 μm, n = 3, p = 0.41). Similarly, the cilia length was uncorrected by the overexpression of EGFP-PACT and EGFP-RABEP2-PACT in siSDCCAG8 cells (13.36 ± 2.194 μm, n = 3 vs. 14.58 ± 2.083 μm, n = 3, p = 0.72). Error bars, SEM.