Confocal 810

Cells were cultured in 4-well chamber slides (LabTek). After serum starvation, cells were washed twice with PBS and fixed in PFA for 10 min and then permeabilized with 0.1% Triton-X-100 in PBS for 15 min at room temperature. Cells were then blocked in 10% goat serum for 1 h at room temperature. Primary antibodies were diluted in PBS containing 1% serum and incubated overnight at 4 °C. Antibodies used were as follows: ARL13B (Proteintech, 1:100); acetylated α-tubulin (Sigma T6793, 1:2000); pericentrin (Abcam, 1:2000). DAPI was used to stain the nuclei. Detection was performed with secondary Alexa Fluor 488/568 antibodies (Invitrogen, 1:1000). Images were captured on Zeiss Confocal 810. Images were collected with 1024 × 1024 pixel definition and Z-sections were taken at 0.5-µm step size. Max projections of the Z-stacks were used for primary cilium counting in ImageJ (NIH).

Cells were cultured in 4-well chamber slides (LabTek). After serum starvation, cells were washed twice with PBS and fixed in PFA for 10 min and then permeabilized with 0.1% Triton-X-100 in PBS for 15 min at room temperature. Then cells were blocked in 10% goat serum for 1 hr at room temperature. Primary antibodies were diluted in PBS containing 1% serum and incubated overnight at 4 °C. Antibodies used were: ARL13B (Proteintech 1:100); anti-acetylated α-tubulin (Sigma T6793 1:2,000); Pericentrin (Abcam 1:2000); DAPI was used to stain nucleus. Detection was performed with secondary Alexa Fluor 488/568 antibodies (1:1000; Invitrogen). Images were captured on Zeiss Confocal 810. Images were collected with 1024 × 1024 pixel definition and Z-sections were taken at 0.5 μm step size. Max projections of the Z-stacks were used for primary cilium measurement and counting in ImageJ (NIH). Cilia length was measure in pixels (px) and compared from at least two experimental replicates with control chondrocytes (N = 226, 226, 67 cilia counted in control, R98-443, and rescued cells).