5 fluorouridine

Immunofluorescence and [³⁵S]-amino acid labeling were performed as previously described (Burgess and Mohr, 2015 (link)). To label nascent rRNA, cells were incubated with 2 mM 5-fluorouridine (Sigma; F5130-100MG) for 20 min. prior to fixation.

FUrd labeling of cells was performed as described previously (74 (link)). After WT or shUBF72 cells were grown to confluency at 10% serum and then for an additional 12 h in the selected serum conditions (0.5%, 10%, and 20%; see Section Cell Culture), they were incubated for 15 min with fresh medium containing 2 mM 5-fluorouridine (Sigma F5130). Immunofluorescence was performed as described using an anti-BrdU primary antibody. Quantitative analysis of captured images, measuring the average mean intensity of the nucleolar signal in each cell from three independent experiments (n = 300 cells in total; 50 cells per condition), was carried out using Imaris (Bitplane AG, v.9.2.1).

10,000 cells were plated onto a 12 mm glass cover slip in a 24-well plate and incubated for 2 nights. The cells were treated with 1 mM AICAR for 12 hrs and then labeled with 2 mM 5-fluorouridine (Sigma) for 15 mins in the incubator. The coverslips were washed with PBS and fixed in 2% formaldehyde (Thermo Fisher Scientific Inc.) for 20 mins in the dark at room temperature. The fixed cells were permeabilized with methanol (Thermo Fisher Scientific Inc.) at −20°C for 10 mins. Coverslips were then blocked with 2% goat serum (Sigma) for 30 mins and incubated with the anti-BrdU antibody (Sigma) at 4°C overnight. The coverslips were washed with PBS and incubated with a secondary goat anti-mouse IgG/IgM (H+L) AlexaFluor^® 488 conjugated antibody (Life Technologies) for 1 hr at room temperature. After washing with PBS 3 times, cells were mounted using the Prolong Diamond anti-fade reagent with DAPI (Life Technologies). Images were obtained as single optical slides using a LSM510-Meta confocal microscope (Zeiss, Jena, Germany) equipped with a 63×1.3 oil immersion objective and excitation wavelengths of 405 nm and 488 nm.

Prior to SMLM experiments, A431 cells were grown on clean #1.5 coverslips for 12–18 hours. The cells were then washed with PBS and fixed with 4% paraformaldehyde for 15 minutes at 4°C. We then mounted the cells on clean slides using mowiol containing DABCO and 50–100 mM MEA, pH 8.5. Before microscopy, the mowiol was allowed to harden for 12–18 hours. The mowiol was freshly prepared according to standard procedures.
For labeling of transcription sites in the cell nucleus, HeLa cells were grown on #1.5 coverslips for 12–18 hours, then incubated for 5 minutes with 5-fluorouridine (Sigma) at a concentration of 10 μM. The cells were then fixed in 2% formaldehyde, permeabilized with 0.1% Triton X-100, and labeled using a mouse monoclonal anti-BrdU antibody (clone BU-33, Sigma). The anti-BrdU antibodies were then detected with a secondary antimouse antibody labeled with Alexa 532 (Invitrogen). The cells were mounted using freshly prepared mowiol containing DABCO and 50–100 mM MEA. Before microscopy, the mowiol was allowed to harden for 12–18 hours.

Kanamycin sulfate, isopropyl β-D-1-thiogalactopyranoside (IPTG), 100X MEM vitamin mixture (for bacterial culture work), bovine serum albumin (BSA), Bromophenol Blue, 5-bromouracil (BrU), 5-bromouridine (BrUrd), 3-(trimethylsilyl)-1-propanesulfonic acid-d₆ sodium salt (DSS-D₆), 5-fluorouracil (FU), 5-fluorouridine (FUrd), nitroblue tetrazolium (NBT), riboflavin, uracil (U) and uridine (Urd) were purchased from Sigma (Oakville, Canada). 5-chlorouracil (ClU) was obtained from Ark Pharm Incorporated (Arlington Heights, USA). The trifluridine (F3TDR) was purchased from Oxchem Corporations (Wood Dale, USA). The BLUelf prestained protein ladder was purchased from FroggaBio (Concord, Canada). The Luria Broth (LB, Invitrogen), Coomassie G-250, HPLC grade H₂O and the Spectra/Por 6–8 kDa molecular weight cut-off (MWCO) dialysis tubing were purchased from Fisher Scientific (Ottawa, Canada). The 3.5 kDa MWCO Snakeskin dialysis tubing was purchased from Thermo Fisher Scientific (Whitby, Canada). The ¹⁵NH₄Cl was purchased from Cambridge Isotope Laboratories (Andover, USA). The MAXYMum Recovery 1.5 mL centrifuge tubes were purchased from Corning (Fairport, USA). The 3 mM SampleJet NMR tubes were obtained from Bruker (Milton, Canada).