Fig. 1

ARL13B-KO RPE1 cells have no cilia. (a) A schematic diagram of CRISPR/Cas9 strategy to make frameshift mutations in human ARL13B gene. The scheme of the exon organization of the human ARL13B gene is illustrated at the top. The middle shows parts of the WT genomic sequences of exon 1 and exon 5, where sequences of two sgRNAs, sg1 and sg2, are highlighted in light blue. Underlined sequences are protospacer-adjacent motifs or PAMs. The bottom shows genomic sequences at sgRNA target sites of two alleles in sg1#a and sg2#4 cell lines. Sequences are aligned with the corresponding WT ones. Inserted and deleted bases are colored red and indicated by “−”, respectively. “+2” and “− 7” indicate insertion of 2 and deletion of 7 bases, respectively. (b) Western blotting of endogenous ARL13B in parental and KO RPE1 cells. Cell lysates from indicated cell lines were subjected to 12% SDS-PAGE followed by immunoblotting using anti-ARL13B and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (loading control) antibodies. Molecular weight markers are labeled to the right of the immunoblot. (c) Fluorescence imaging of endogenous ARL13B and acetylated tubulin in parental and KO RPE1 cells. Indicated cells were fixed and co-immunostained by antibodies against indicated cilium markers before being subjected to wide-field microscopy. (d) Quantification of the ciliogenesis percentage in parental and KO RPE1 cells. Images acquired as described in (c) were analyzed. n = 3 independent experiments with ≥ 100 cells analyzed in each case. (e) Fluorescence imaging of endogenous glutamylated tubulin and pericentrin in parental and KO RPE1 cells, similar to (c). (f) Silent mutations in ARL13B^R-GFP make it resistant to sgRNAs. Regions of the ARL13B^R coding sequence, corresponding to sg1, sg2, sg4, and sg5 (colored blue), are shown with introduced multiple silent mutations colored red. Protospacer-adjacent motifs, or PAMs, are underlined. Superscript numbers indicate base pair positions in the coding sequence. (g) ARL13B^R-GFP can rescue the no-cilium phenotype of ARL13B-KO cell lines. Parental RPE1, sg1#a, and sg2#4 cells expressing ARL13B^R-GFP were processed for immunofluorescence to label endogenous acetylated tubulin and pericentrin. Arrows indicate ciliated cells. In (c, e, and g), the boxed region is enlarged at the lower left corner, and the scale bar represents 10 μm. (h–j) ARL13B^R-GFP expressing sg1#a cells exhibit normal cilia. Images acquired as described in (g) were used for quantification. Cilia were identified by acetylated tubulin. Panel h shows the ciliogenesis percentage in GFP-positive parental and sg1#a cells (indicated by “+”). It also displays the ciliogenesis percentage of GFP-negative sg1#a cells from the same coverslips (indicated by “−”). Quantification was from n = 3 independent experiments with ≥ 100 cells analyzed in each experiment. In (i), the cilium length was measured in ciliated GFP-positive cells. Quantification was from n = 3 independent experiments with ≥ 30 cells analyzed in each experiment. In (j), the CPIR of ARL13B^R-GFP was quantified in ciliated GFP-positive cells. Quantification was from n = 3 independent experiments with ≥ 30 cells analyzed in each experiment. In (d, h–j), the error bar represents the mean ± standard deviation.