Plan apochromat oil immersion objective

Cells were fixed (3% paraformaldehyde, 15 min room temperature), permeabilized using Triton X-100 (0.1%), blocked with 2% BSA (in PBS) and stained with mouse anti-Arl13B and goat anti-mouse Alexa 555 antibodies (Both 1:1000). Nuclei were stained with DAPI (Sigma). Coverslips were mounted with Mowiol and NIH3T3 cilia visualized using an Olympus IX70 microscope with 60 × 1.4 NA Plan-Apochromat oil immersion objective (Olympus, Center Valley, PA) and a charge-coupled device camera (CoolSNAP HQ, Photometrics, Tucson, AZ). MEF-R1441G cells were visualized using an SP8 laser scanning confocal microscope (Leica Microsystems, Germany) using a 63 × 1.4 NA oil immersion objective. When needed, maximum intensity projections were made and images analyzed using ImageJ.

HeLa control and Nup35 knockdown cells were fixed and stained with rabbit anti-Nup155 (Abcam) and mouse anti-Nup153 (Abcam) at room temperature for 1 h and incubated with goat anti-rabbit Alexa Fluor 488 (Molecular Probes) and goat anti-mouse Alexa Fluor 546 (Molecular Probes) at room temperature for 1 h. Subsequently, samples were incubated for 20 min at room temperature with directly conjugated rabbit anti-Lamin B1 antibody, which was labeled using Zenon^TM Alexa Fluor^TM 647 Rabbit IgG Labeling Kit (Zenon) according to the manufacturer’s instructions. 3D SIM imaging was performed on a DeltaVision OMX SR microscope (GE Healthcare) equipped with a 60× 1.40 numerical aperture Plan Apochromat oil immersion objective (Olympus). Samples were mounted with ProLong Gold Mounting Medium (Invitrogen) and image stacks with 15 images per plane (five phases, three angles) and a z-distance of 125 nm were captured and then subjected to a computational reconstruction (softWoRX; Version 6.1, Applied Precision).

Immunofluorescence analysis and confocal microscopy analysis were performed as previously described [31 (link)]. When indicated, 1 µM cytochalasin D, latrunculin A, jasplakinolide, paclitaxel, 10 µM nocodazole, or 200 µM NSC23766 were added to CHO-K1/ACKR2 cells 1 h before being incubated with chemokine, while 1 μM staurosporine was added 6h before. When PTX was used, cells were treated with 100 ng/mL for 16 h before adding chemokine. To detect microtubules, cells were incubated for 1 h at RT with 1 μg/mL anti-α-tubulin. To visualize myosin Vb and Rab11, cells were incubated for 1 h at RT with 2.5 μg/mL anti-myosin Vb and anti-Rab11, respectively. High-resolution images (1024 × 1024 pixels) were acquired sequentially with a 60x/1.4 numerical aperture Plan-Apochromat oil immersion objective on a FV1000 laser scanning confocal microscope (Olympus, Shinjuku, Tokyo, Japan) and assembled using Photoshop software (Adobe Systems, San Jose, CA, USA). Pearson’s coefficient of correlation (PCC) was measured in a selected region of interest representative of the analyzed cell using Imaris-Coloc software (Olympus, Shinjuku, Tokyo, Japan).