Figure 3.
LUZP1 and EPLIN are novel negative regulators of ciliogenesis. (A) LUZP1 depletion increases ciliation in RPE-1 cells. IF analysis of RPE-1 cells stably expressing siRNA-sensitive or resistant GFP-LUZP1 and transfected with control (NTsiRNA) or siRNAs targeting LUZP1 for 72 h. The cells were stained for γ-tubulin (centrosome and basal body marker) and ARL13B (ciliary marker). DNA was stained with DAPI. The arrowheads indicate primary cilia. (B) Bar graph shows the mean percentage of ciliated cells (n > 200 cells per sample, three independent experiments) in both RPE-1 stable lines transfected with the indicated siRNAs for 72 h. Error bars indicate SD. **, P < 0.01 (Student’s two-tailed t test). (C) Western blot analysis of endogenous and GFP-tagged LUZP1 in both stable lines transfected with control nontargeting (NT) or LUZP1-directed siRNAs for 72 h. (D) EPLIN silencing increases ciliation in RPE-1 cells. IF analysis of RPE-1 cells stably expressing GFP-only or GFP-EPLINα and transfected with control (NT siRNA) or siRNAs targeting the 3′ UTR of EPLIN for 72 h. The cells were stained for γ-tubulin (centrosome and basal body marker) and ARL13B (ciliary marker). DNA was stained with DAPI. The arrowheads indicate primary cilia.(E) Bar graph shows the mean percentage of ciliated cells (n > 200 cells per sample, three independent experiments) in both stable lines transfected with the indicated siRNAs for 72 h. Error bars indicate SD. *, P < 0.05 (Student’s two-tailedt test). (F) Western blot analysis of endogenous and GFP-tagged EPLINα in the cells transfected with control nontargeting (NT), or LUZP1-directed siRNAs for 72 h. (G and H) LUZP1 and EPLIN depletion leads to increased ciliary length in RPE-1 cells. The graphs show the length of cilia measured in the indicated populations treated wither with control siRNA or siRNAs directed against LUZP1 or EPLIN.n > 200 cilia per condition (data pooled from three independent experiments). Bars correspond to the average and SD. ***, P < 0.001 (Student’s two-tailed t test); ns, nonsignificant.

LUZP1 and EPLIN are novel negative regulators of ciliogenesis. (A) LUZP1 depletion increases ciliation in RPE-1 cells. IF analysis of RPE-1 cells stably expressing siRNA-sensitive or resistant GFP-LUZP1 and transfected with control (NTsiRNA) or siRNAs targeting LUZP1 for 72 h. The cells were stained for γ-tubulin (centrosome and basal body marker) and ARL13B (ciliary marker). DNA was stained with DAPI. The arrowheads indicate primary cilia. (B) Bar graph shows the mean percentage of ciliated cells (n > 200 cells per sample, three independent experiments) in both RPE-1 stable lines transfected with the indicated siRNAs for 72 h. Error bars indicate SD. **, P < 0.01 (Student’s two-tailed t test). (C) Western blot analysis of endogenous and GFP-tagged LUZP1 in both stable lines transfected with control nontargeting (NT) or LUZP1-directed siRNAs for 72 h. (D) EPLIN silencing increases ciliation in RPE-1 cells. IF analysis of RPE-1 cells stably expressing GFP-only or GFP-EPLINα and transfected with control (NT siRNA) or siRNAs targeting the 3′ UTR of EPLIN for 72 h. The cells were stained for γ-tubulin (centrosome and basal body marker) and ARL13B (ciliary marker). DNA was stained with DAPI. The arrowheads indicate primary cilia.(E) Bar graph shows the mean percentage of ciliated cells (n > 200 cells per sample, three independent experiments) in both stable lines transfected with the indicated siRNAs for 72 h. Error bars indicate SD. *, P < 0.05 (Student’s two-tailedt test). (F) Western blot analysis of endogenous and GFP-tagged EPLINα in the cells transfected with control nontargeting (NT), or LUZP1-directed siRNAs for 72 h. (G and H) LUZP1 and EPLIN depletion leads to increased ciliary length in RPE-1 cells. The graphs show the length of cilia measured in the indicated populations treated wither with control siRNA or siRNAs directed against LUZP1 or EPLIN.n > 200 cilia per condition (data pooled from three independent experiments). Bars correspond to the average and SD. ***, P < 0.001 (Student’s two-tailed t test); ns, nonsignificant.

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