Figure 5. Mib1 is downstream of PCM1.

(A) Validation of siRNA knockdown of Mib1. Control (sgCTL), PCM1, and TALPID3 KO RPE1 cells were transfected with the indicated siRNAs corresponding to non-specific control (siGL2) or Mib1 (siMIB1-1 or siMIB1-2), and lysates were subjected to western blot analysis with the indicated antibodies. (B) Mib1 depletion partially rescues the ciliogenesis defect in PCM1 KO cells. Cell lines in panel A were transfected with siRNAs corresponding to non-specific control (siGL2) or Mib1 (siMIB1-1 or siMIB1-2) for 48 hr and serum starved for 48 hr. Ciliated cells (n ≥ 100 per sample in three independent experiments) were analyzed by immuno-staining with GT335. Error bars, SEM. *p<0.05. (C) E3 ligase activity of Mib1 underlies ciliogenesis defect in PCM1 KO cells. Control and PCM1 KO RPE1 cells were infected with empty (EV), Flag-Mib1-WT or Flag-Mib1-C985S lentiviruses for 72 hr and serum starved for 48 hr. Ciliated cells were analyzed by immuno-staining with GT335; n≥100 per sample were analyzed in two independent experiments. Error bars, SD. *p<0.05. (D) Control, PCM1, and Talpid3 KO RPE1 cells were transfected with siGL2, siMIB1-2, or siMIB1-3 for 48 hr and subjected to western blot analysis with the indicated antibodies.

DOI: http://dx.doi.org/10.7554/eLife.12950.010

Figure 5—figure supplement 1. Mib1 suppress ciliogenesis.

(A) RPE1 cells were transfected with siCTL, siMIB1-2 or siMIB1-3 for 2 days. The ciliated cells were determined by immunostaining with GT335 antibody. N ≥ 100 per sample in three independent experiments. Error bars, SEM. *p<0.05. (B) RPE1 cells were infected with empty, Flag-Mib1-WT, or Flag-Mib1-C985S lentiviruses for 72 hr and serum starved for 48 hr. GT335-positive ciliated cells were counted. N ≥ 100 per sample in two independent experiments. Error bars, SD. *p<0.05. (C) and (D) RPE1 cells were transfected with siCTL, siMIB1-2 or siMIB1-3 for 2 days and immunostained with GT335 (green) and Ki-67 (red) antibodies. The ciliated cells were counted in Ki-67 (C) positive cells or (D) negative cells. N ≥ 100 per sample in two independent experiments. Error bars, SD. *p<0.05. (E) Mib1 depletion partially rescues the ciliogenesis defect in PCM1 KO cells. Control (sgCTL), PCM1, and TALPID3 KO RPE1 cells were transfected with the indicated siRNAs corresponding to non-specific control (siGL2) or Mib1 (siMIB1-1 or siMIB1-2) for 48 hr and serum starved for 48 hr. Ciliated cells were analyzed by immuno-staining with GT335.

DOI: http://dx.doi.org/10.7554/eLife.12950.011

Figure 5—figure supplement 2. Generation of TALPID3 knock-out cell lines.

(A) Gene-editing with CRISPR/Cas9 was used to create the indicated mutations in RPE1 cells, the sequence for which is shown. (B) Talpid3 protein was undetectable by immunofluorescent detection and western blotting. Higher magnification views of centrioles are shown below the main panel in (B). Scale bar, 5 µm. (C) localization of PCM1 and Mib in TALPID3 KO cells. Control and TALPID3 KO RPE1 cells were incubated with or without NZ (1 μg/ml) for 8 hr. Cell were then immuno-stained with PCM1 (green), Mib1 (green), Centrin (red) and GT-335 (red). Scale bar, 2 µm.

DOI: http://dx.doi.org/10.7554/eLife.12950.012

Figure 5—figure supplement 3. Mib1 regulates the stability of multiple centriolar satellite proteins.

(A) and (B) RPE1 or U2OS cells were infected with empty, Flag-Mib1-WT, or Flag-Mib1-C985S lentivirus for 3 days and subjected to (A) western blotting with the indicated antibodies or (B) immunostaining with DAPI (blue), GT335 (red), and Cep131 (green), CEP290 (green) or PCM1 (green). Scale bar, 5 µm. N ≥ 100 per sample in two independent experiments. Error bars, SD. *p<0.05. (C) and (D) RPE1 cells were transfected with siRNA for Mib1 and (C) immunostained with DAPI (blue), centrin (red), and Cep131 (green), CEP290 (green) or PCM1 (green) or subjected to (D) western blotting with the indicated antibodies.

DOI: http://dx.doi.org/10.7554/eLife.12950.013