Poly l lysine precoated glass coverslips

All cell imaging experiments were performed using a Nikon Eclipse 80i epifluorescence microscope (Melville, NY) with a 60× 1.40 oil objective, and a Hamamatsu ORCA ER digital camera (Houston, TX) was used to acquire images. The MDA-1986 cells were seeded onto poly-L-lysine precoated glass coverslips (BD, Franklin Lakes, NJ) in 12-well culture plates at a density of 50,000 cells per well and allowed to grow overnight. Subsequently, cells were treated with PPa (6.48 μg/ml) or HA-ADH-PPa (6.48 ug/ml of PPa) in 1:5:94 (v/v/v) TWEEN® 80/DMSO/media followed by incubation at 37 °C for 5 h. The cell nuclei and lysosomes were stained with DAPI (10 μg/ml) for 5 min and LysoTracker® Blue DND-22 (4 μM) for 30 min, respectively. After three washes with 3 ml of PBS, the coverslips were placed on the slide glasses for imaging. The live cells were immediately imaged using a yellow fluorescent protein (YFP, ex/em, 500/535 nm) filter set (Nikon, NY) for imaging PPa and Ultraviolet excitation (UV-2E/C, ex/em, 360/400 nm) filter set (Nikon, NY) for imaging DAPI and LysoTracker® Blue DND-22 separately.

For Gpr161, Smo or Gli2 cilia localization assays, IMCD3, NIH-3T3, Smo-GFP, Smo-M2-myc, or SmoL412F-myc cells were grown to confluence on 12mm poly-L-Lysine pre-coated glass coverslips (BD Biosciences). The indicated compounds were dissolved in DMEM/3%CS and then applied to cells for 48hrs or 72hrs. Cells were either fixed in 100% methanol for 5min at −20°C or in 3.7% formaldehyde for 15min at room temperature. The cells were further permeabilized in blocking buffer (5% normal goat serum and 0.2% Triton X-100 in PBS) for 10min. Coverslips were treated with anti-Smo (provided by P. A. Beachy), anti-Myc (Cell Signaling Technology, 2272), anti-GFP (MBL International, 598), anti-Gli2 and anti-Gpr161 (provided by S. Scales, Genentech), and anti-tubulin (Sigma, T6793) in blocking buffer each for 30min. After several PBS washes, the coverslips were further incubated with Alexa Fluor 488-conjugated goat anti-rabbit, Alexa 594-conjugated goat anti-mouse, or Hoechst 33342 (Invitrogen) for 30min. 100 primary cilia were scored for the presence of Gpr161, Smo, SmoM2, SmoL412F, and Gli2 in each experiment.

MDCK-MDR cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) with 1× l-glutamine and sodium pyruvate that was supplemented with 10% FBS, 1× non-essential amino acid, and 1× penicillin-streptomycin. The cells were seeded onto poly-l-lysine precoated glass coverslips (BD, Franklin Lakes, NJ) in a 12-well culture plate at a density of 5000 cells/well.