Deltavision spectris fluorescence microscope

All cell images were recorded using the DeltaVision Spectris fluorescence microscope (Applied Precision). Live cell images were prepared as previously described (http://www.lamondlab.com/f7protocols.htm). Cells were imaged using a 60x (NA 1.4) Plan Apochromat objective. Twelve optical sections separated by 0.5 μm were recorded for each field and each exposure (SoftWoRx image processing software, Applied Precision). Primary antibodies against SMN1 (BD Transduction Laboratories), fibrillarin (72b9) [31 (link)], Dyskerin (Sigma-Aldrich) and coilin (5P10) [32 (link)] were used for immunostaining. Primary antibodies against HIF-1α (610958, BD Biosciences), HIF-1β (3718, Cell Signaling), CAIX (NB100-417, Novus Biologicals), PHD2 (Bethyl A300-322A), BNIP3 (ab10433, Abcam), PHD3 (A300-327A, Bethyl labs) and Actin (3700, Cell Signaling) were used for immuno blotting analysis.

FISH procedure was performed as previously described [http://www.singerlab.org/protocols]. HeLa cells were transfected with a plasmid vector containing the snoMEN mini gene expressed from the CMV promoter. The cells were fixed with 4% paraformaldehyde after pre-permeabilization with 1% tritonX-100. After 70% ethanol treatment, a Cy-3 labeled snoMEN specific oligonucleotide probe (CM1: 5’-GCAGCACGACUUCUUCAAGUC-3’, CM2: 5’-GCAGAAGAACGGCAUCAAGGU-3’ CM3: 5’-CAGCCACAACGUCUAUAUCAU-3’) was hybridized with Fluorescein labelled U3 specific oligonucleotide probe (5’-ACCACUCAGACCGCGUUCUCUCCC-3’) as a nucleolar marker, using standard procedures. The fluorescence signal was imaged using a Deltavision Spectris fluorescence microscope (Applied Precision). The specific probes for each of the snoRNAs (U47: 5’-UGGAACGGUUUUACAGUGAUAUC-3’, U77: 5’-ACAUCUCUUCAUGAUUAAAUcugcug-3’, ACA16: 5’-ACCGUCAAGGAAAACUGUCACUCUGGGC-3’, miR-566: 5’-GUUGGGAUCACAGGCGCCC-3’) were also labelled with Cy-3.

All cell images were recorded using the DeltaVision Spectris fluorescence microscope (Applied Precision). Cells were imaged using either a 10X, or 60X (NA 1.4), Plan Apochromat objective lens. Twelve optical sections, each separated by 0.5 μm, were recorded for each field and each exposure (SoftWoRx image processing software, Applied Precision). Primary antibodies against Fibrillarin (72b9)[47 (link)], Tubulin (DM1A, Sigma), Cleaved Caspase-3 (Asp175, Cell Signalling Technology), Cleaved PARP (Asp214, Cell Signalling Technology), SMN1 (BD Transduction Laboratories), Ago1 (D84G10, Cell Signalling Technology), Ago2 (C34C6, Cell Signalling Technology), Upf1 (Cell Signalling Technology) and Lamin A/C (636 Santa Cruz), were used for immunostaining and/or western blotting.