Cell culture, transfection, immunocytochemistry, and imaging

Grow hTERT-RPE1 cells in DMEM/F12 (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% glutaMAX, 1% penicillin-streptomycin. Keep cells at 37°C with 5% CO2 in an enclosed incubator.

For live-cell imaging experiments, seed the cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 1-2 × 10⁵ cells.

For RPE1: allow the cells to adhere for8h 0m 0s-24h 0m 0sbefore being transiently transfected using 4µL Lipofectamine™ 2000 Transfection Reagent (Invitrogen) in Opti-MEM media, mix them with the respective plasmids (1µg -2µg) and visualize after 48h 0m 0s.

For cilia generation, serum-starve the cells in DMEM/F12 media (without FBS) for 48h 0m 0s.

For live-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 3-5 × 10⁵ cells.

Use2.4µL of Lipofectamine™ Stem Transfection Reagent (Invitrogen) in Opti-MEM media with respective plasmids (1µg-3µg) and visualize at least 48h 0m 0s later.

For fixed-cell imaging experiments, seed cells on glass-bottom dishes (MatTek; 35mm) at the concentrations ranging from 1-3 x 10⁵cells.

• For immunofluorescence visualization, fix the cells with 4% (v/v) paraformaldehyde (Electron Microscopy Sciences) in 1× phosphate-buffered saline (PBS) for 0h 20m 0s.

Wash cells thrice in PBS.

Perform cell extraction with 0.25-0.5% (v/v) Triton X-100 in PBS for 0h 10m 0s.

Wash cells thrice in PBS.

For removal of free aldehyde groups, quench the cells with fresh1sodium borohydride (Sigma-Aldrich) in PBS for 0h 7m 0s, and then wash thrice in PBS.

Further block the cells for 0h 30m 0s in 5% bovine serum albumin (BSA, Sigma-Aldrich) in PBS and then incubate 0h 10m 0s at 4°C with the respective antibodies listed in Table S1.

Wash the cells with PBS thrice the following day and incubate with Alexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific) for 1h 0m 0s at Room temperature.

Wash the cells thrice in 1×PBS.

Use DAPI (Thermo Fisher Scientific) for nuclear staining, when necessary.

For live imaging, maintain cells in a caged incubator with humidified atmosphere (5% CO₂) at 37°C.