Nucleolin

Fetal bovine serum (FBS) was purchased from Gibco/BRL (Burlington, ON, Canada). Pen Strep was purchased from life technologies corporation, Grand Island, NY, USA. Enhanced chemiluminescence (ECL) regent was purchased from Amersham, Arlington Heights, IL, USA. The enzyme linked immunosorbent assay kit (ELISA) for prostaglandin E2 (PGE2) (cat# KGE004B) and IL-1β (cat# MLB00C) were purchased from R&D System Inc. (Minneapolis, MN, USA). ELISA kits for mouse TNF-α (cat# 560478), and IL-6 (cat# 550950) were purchased from Becton, Dickinson and Company (BD Biosciences; San Jose, CA, USA). Cytosolic and nucleus proteins extracted from commercial protein extraction kit (#78833) were purchased from Thermo Scientific; Rockford, USA. The following primary antibodies for Western blots: iNOS (#13120), COX2 (#12282), phospo NF-κB-P50 (#4806), NF-κB-P50 (#3035), phospo NF-κB-P65 (#3033), NF-κB-P65 (#8242), nucleolin (#14574), β-actin (#4970), and rabbit secondary antibody (#7074) were purchased from Cell Signaling Technology MA, USA). All analytical grade organic solvents were purchased from Sigma-Aldrich or otherwise mentioned in the text.

All reagents used in this study [chlorpromazine, sucrose, methyl-β-cyclodextrin, nystatin, calcium chloride, ethylene glycol tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), potassium chloride, magnesium chloride and dithiothreitol (DTT)] were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit and affinity-purified polyclonal anti-Cpne7, and anti-Dsp antibodies were produced as described previously^{46 (link), 47 (link)}. Commercial antibodies against Flag (Sigma-Aldrich), nucleolin (Cell Signaling; Danvers, MA, USA), Dmp1 (Abcam; Cambridge, UK), glyceraldehyde 3-phosphate dehydrogenase (Gapdh) (Santa Cruz Biotechnology; Santa Cruz, CA, USA), α-tubulin (Santa Cruz Biotechnology), and Ift-88 (Santa Cruz Biotechnology) were used for Western blot analyses. Expression vectors encoding DDK (Flag)-tagged Cpne7 (NM_153636) and recombinant Cpne7 (NP 705900) were purchased from Origene (Rockville, MD, USA). Control siRNA and nucleolin-targeting siRNA were purchased from Ambion (Carlsbad, CA, USA). Ift88 siRNA was purchased from Santa Cruz Biotechnology.

To measure pre-rRNA biosynthesis, cells were seeded at 50% confluence in six-well plates containing round cover glasses (12CIR-1D; Thermo Fisher Scientific). After treatment, cells were incubated with 10 µM 5-Fluorouridine (5FU) (TCI chemicals) for 3 h, followed by fixation with methanol at −20 °C for 30 min and blocking with milk (5% in PBS). 5FU was visualized using primary BrdU antibody (1:500 Sigma-Aldrich) and the nucleolus was visualized by staining for nucleolin (1:1000 Cell signaling technology). Detection of primary antibodies was performed with Alexa Fluor 488 Goat anti-Mouse IgG (1:2000 Thermo Fisher Scientific) and Alexa Fluor 594 Goat anti-Rabbit IgG (1:2000 Thermo Fisher Scientific). Slides were mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and viewed using BZ-X710 fluorescence microscope or LSM700 confocal microscope (CLSM, Carl Zeiss, Jena, Germany). For 5FU fluorescence quantification, three images were taken at random per treatment with a 100× objective lens and signal intensity was quantified from using ImageJ software. 5FU intensity for each image was divided by the number of cells and fold change was calculated by setting the control cells to one.

Cells were lysed in mammalian protein extraction reagent (Pierce). Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce), and equal amounts of proteins (30 µg) were separated by 10% SDS–PAGE under denaturing conditions and transferred to PVDF membranes (Millipore). Membranes were blocked in 5% non-fat milk (Bio-Rad) and then incubated with primary antibodies, followed by incubation with a secondary antibody conjugated with horseradish peroxidase (HRP, 1:10,000; Amersham Biosciences). Immunoreactive proteins were visualized using the LumiGLO chemiluminescent substrate (Cell Signaling Technology). The primary antibodies are as follows: β-Actin (1:10,000; Sigma); α-Tubulin (1:5000; Sigma); LIN28B (1:1000; Cell Signaling Technology, #11965); TP53 (1:1000; Cell Signaling Technology, #2527); RPL26 (1:1000; Abcam, ab59567); cleaved caspase-3 (1:1000; Cell Signaling Technology, #9664); Nucleolin (1:1000; Cell Signaling Technology, #14574); MDM2 (1:1000; Cell Signaling Technology, #51541); MDM4 (1:1000; Sigma-Aldrich, 04-1556); and LaminB1 (1:1000; Abcam, ab16048).

U2OS cells were seeded on chamber slides. A day after seeding, cells were washed twice with PBS and permeabilized with 0.5% Triton X-100 for 5 min on ice before fixation with 4% paraformaldehyde for 15 min at RT. After washing with PBS, 0.5% Triton X-100 was added to permeabilize the cells. For chromatin-bound protein analysis, this series of steps was altered as follows: permeabilization with 0.5% Triton X-100 for 2 min and fixation with 100% methanol for 30 min. Then, cells were treated with blocking buffer for 1 h and incubated with primary antibodies overnight. After washing, cells were incubated with Alexa Fluor-conjugated secondary antibodies for 1 h. PBS-washed cells were visualized with a fluorescence microscopic imaging system. Antibodies against S9.6 (Kerafast, Cat # ENH001), Nucleolin (Cell Signaling Technologies, Cat# 14574), γH2AX (Millipore, Cat# 05–636), and 53BP1 (Cell Signaling Technologies, Cat# 4937) were used.