Fig 2: MT Nucleation from the NE Requires Nesprin-1, Sun1/2, and Akap450(A) 3D-SIM fluorescent image of the nucleus of a differentiated C2C12 cell, stained for Nesprin-1 (white, MANNES1E), Pericentrin (Pcnt, red), and microtubules (MTs, green) after 5 min nocodazole washout to allow MT regrowth. The scale bar represents 5 µm.(B) 48 hr differentiated C2C12 cells, treated with the indicated siRNAs, were immunostained for Pericentrin (Pcnt, red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. The scale bar represents 10 µm. See also Figure S3A.(C) Quantification of the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (B). Error bars ± SD; n represents total number of nuclei from at least three independent experiments. ***p < 0.001; **p < 0.01; n.s., not statistically significant, t test.(D) Differentiated human immortalized myotubes from a healthy control (wild-type) or from a patient carrying a nonsense mutation within the SYNE1 gene (23560 G>T) were immunostained for PCM1 (red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. Images represent maximum projections of confocal z sections. The scale bar represents 10 µm. See also Figures S3B and S3C.(E) Quantification of the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (D). Error bars ± SD; n represents total number of nuclei from two independent experiments. **p < 0.01; t test.(F) 48 hr differentiated C2C12 cells, treated with the indicated siRNAs were immunostained for Akap450 (red), microtubules (MTs, green) and Myogenin (MYOG, gray) after nocodazole washout. The scale bar represents 10 µm.(G) Graph shows the mean percentage (%) of Myogenin-positive cells with MT nucleation from the NE as described in (F). Error bars ± SEM; n represents total number of nuclei from two independent experiments. *p < 0.05; n.s., not statistically significant, one-way ANOVA with Dunnett’s multiple comparisons test.

Fig 3: AZI1 interacts with the BBSome through BBS4. A) A silver stained gel from GFP IP sample of testis lysate. Lane 1 shows the wild-type (WT) precipitate and lane 2 is the precipitate of testis lysate from transgenic (TG) animals. The band labeled with an asterisk is the 120 KDa band identified as Azi1 by mass spectrometry. B) Western blot showing interaction of BBS4 with AZI1 and PCM1. FLAG and S tagged BBSome subunits were transfected in 293T cells and Co-IP was performed using FLAG agarose beads. The top blot shows efficient transfection of the BBS subunits detected by anti-FLAG antibody. The lower two blots were immunoblotted with AZI1 and PCM1 antibody, respectively. C) FLAG-BBS4 stable cell (293T) was co-immunoprecipitated with FLAG agarose beads. Input, supernatant and eluate were immunoblotted with, antibody against GAPDH to show that equal amounts were loaded, antibody against FLAG to show efficient precipitation of BBS4 by FLAG beads, and antibody against AZI1 to show amounts of AZI1 precipitated by BBS4 in our cell line. D) 293T cells stably expressing the FLAG tagged BBSome subunits BBS4, 5, 8, and 9 were co-IPed with FLAG agarose beads and immunoblotted with AZI1 antibody. Although all the subunits were able to precipitate AZI1, BBS4 showed the most efficient pull-down. E) Co-IP using AZI1 antibody was performed in 293T cells followed by Western blotting with different BBS antibody to show interaction of endogenous BBS proteins and AZI1.

Fig 4: AZI1 knockdown reduces ciliogenesis but increases ciliary localization of the BBSome. A) RPE-1 cells were transfected with siRNA against BBS4, PCM1, and AZI1. Approximately 500 cells per sample were counted. B) AZI1 depletion increases the ciliary localization of BBS9 compared to the control knockdown. Red staining represents BBS9, and cilia are stained with acetylated α-tubulin. Nuclei are stained blue with DAPI. C) Graph showing a significant increase in cells with ciliary BBS9 upon AZI1 knockdown by different siRNAs. D) BBS8 (red) is used as a BBSome marker to confirm increased ciliary localization of the BBSome upon AZI1 knockdown. Cilia (green) in the insets of figures B and D are slightly shifted to show ciliary localization of BBS proteins. E) The graph shows a significant increase in the number of ciliated cells with BBS8 or BBS9 upon AZI1 knockdown, and decrease in ciliary BBS8 or BBS9 localization upon AZI1 overexpression. Approximately 250 ciliated cells were counted in control as well as AZI1 knockdown culture of RPE-1 cells. All data are presented in mean +/− SEM. Significance is calculated using Student's t-test for C and E, and one way ANOVA for A. P<0.05 is considered significant for each analysis.

Fig 5: BBS4 is part of a PCM1 dependent centriolar satellite complex.293T cells were transfected with siRNA against CTRL, AZI1, and PCM1, and the cell lysates were separated by 10–40% sucrose gradient centrifugation and fractionation. Thirteen fractions, (from 7–19, 19 being the heaviest fraction) were run on an SDS-PAGE gel and analyzed by immunoblotting with antibodies against various BBS proteins, AZI1, and PCM1. Fractions marked red represent the peak for the BBSome. Fractions marked blue is the peak for centriolar satellite pool of BBS4, along with centrosomal proteins, AZI1 and PCM1.