Fibrillarin

Cells were lysed, and immunoblotting was performed as previously described (Bianco and Mohr, 2017 (link)). The antibodies used in this study were as follows: TIF-IA (Bethyl Laboratories; A303-065A) UBF (Santa Cruz; SC-13125), Akt (Cell Signaling; 9272), Fibrillarin (Santa Cruz; sc-166001), UL44 (Virusys; CA006), pp28 (Virusys; CA004-100), IE1/IE2 (Millipore; MAB810), HMGB2 (Cell Signaling Technology; 14163), USP18 (Cell Signaling Technology; 4813), IFIT2 (Proteintech; 12604–1-AP), TFIIIA (Bethyl; A303-621A), ISG15 (Proteintech; 15981–1-AP), RIG-I (Proteintech; 20566), MDA5 (Proteintech; 21775–1-AP), UBA7 (Cell Signaling Technology; 69023), actin (Cell Signaling Technology; 3700), BrdU (Sigma; B2531), p53 (Cell Signaling Technology; 9282).

MDA-MB-231, MDA-MB-453, MDA-MB-468, MCF-7, BT-474 and SK-BR-3 mammary carcinoma cell lines were serum starved in DMEM containing 0.1% bovine serum albumin (BSA, SIGMA) and then stimulated with 10% FBS or 5% WF for the indicated time points.
The preparation of protein lysates and immunoblotting analysis was performed as previously described [33 (link)], except that membranes were blocked with Odyssey Blocking Buffer (Licor, Biosciences) and, following incubation with primary antibodies overnight at 4°C, incubated 1 hour at RT with IR-conjugated (Alexa Fluor 680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, Licor).
Primary antibodies directed against AKT (sc-1618), ERK1 (sc-94), STAT3 (sc-482), Fibrillarin (sc-25397) and Vinculin (sc-7694) were purchased from Santa Cruz Biotechnology, Inc.; pT202/204 ERK1/2 (#9101), pS473 AKT (#4060), pY705STAT3 (#9131) were purchased from Cell Signaling; Tubulin (#T9026) was purchased from SIGMA; Cyclin D1 (#04-1151) and Bcl2 (#OP60) were purchased from Millipore; Grb2 was purchased from BD Transduction Laboratories (#610112).

Harvested cells were resuspended in 20 mM RIPA buffer (pH 7.4) containing a cocktail of proteinase inhibitors (Calbiochem, Merck, Darmstadt, Germany) and treated by sonication (Microson XL-2000, Minisonix, Farmingdale, NY, USA) to obtain a total protein extract. For cytosol-nucleus fractionation, proteins were isolated by using the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem) as suggested by the manufacturer. Proteins were assayed by the BCA Protein Assay (Thermo Scientific, Rockford, IL, USA), analyzed by SDS-PAGE and western blotting as reported elsewhere [40 (link)]. Membranes were probed with primary antibodies against acetyl-H3, acetyl-H4, active-β-catenin, and PP2A (Upstate Biotechnology, Millipore, Bilerica, MA, USA); acetyl-α-tubulin and α-tubulin (Sigma-Aldrich); GAPDH, cleaved PARP, cleaved caspase-9, c-Myc and phospho-GSK-3β(ser9) (Cell Signaling Technology, Danvers, MA. USA); cyclin D1, GSK-3β, I2PP2A, CIP2A and fibrillarin (Santa Cruz Biotechnology); suitable peroxidase-conjugated IgG preparations (Sigma-Aldrich) were used as secondary antibodies; the ECL procedure was employed for development. Western immunoblots reported all through this work were the mean ± SD of experiments carried out in triplicate (unless otherwise specified) and the software ImageJ [41 (link)] was used for densitometric quantification of protein band intensity.

Human fibrosarcoma cell line HT1080 and Mouse Embryonic Fibroblasts (MEFs) cells were grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin–streptomycin solution, at 37 °C under a 5% CO₂ atmosphere. siRNAs were purchased from Qiagen (Hilden, Germany) (RNF4 #1: SI03228512, RNF4 #2: SI04167828). AllStars negative control siRNA (Qiagen) was used as a control. siRNAs were transfected using Lipofectamine RNAiMAX (Merck Millipore, Burlington, VT, USA), according to the manufacturer’s protocol. All reagents were obtained from commercial sources; TNF-α (Enzo Life Sciences, Farmingdale, NY, USA), 5z-7-oxozeaenol(5Z-7), BV-6, necrostatin-1 (Santa Cruz, Dallas, TX, USA), cycloheximide (Sigma, St. Louis, MO, USA). The antibodies used were against caspase-8 (Enzo Life Sciences), RNF4 (Proteintech, Rosemond, CA, USA), FLAG (Sigma), α-tubulin, p65, Fibrillarin (Santa Cruz), caspase-3, phospho-p38, total p38, phospho-JNK, total JNK, phospho-ERK, total ERK, phospho-RIPK1 (Cell signaling, Danvers, MA, USA), total RIPK1 (Becton and Dickinson, Franklin Lakes, NJ, USA), and β-actin (Wako, Tokyo, Japan).

Immunolocalization of proteins and quantification of nucleolar, nuclear and cytoplasmic fluorescence followed published procedures [14] , [43] (link), [44] (link). Immunostaining was performed with antibodies against B23 (Cell Signaling; #3542; diluted 1∶700), fibrillarin (Santa Cruz, sc-25397; 1∶500), nucleolin (sc-13057; 1∶1,000), RPA194 (sc-48385; 1∶200) or lamin A (sc-20680; 1∶500). Using MetaXpress software modules, nucleoli were identified with the Detect light holes filter for B23, fibrillarin or nucleolin, and the Detect dark holes filter for RPA194. The staining pattern obtained for DAPI provided the reference for dark holes. To evaluate de novo DNA synthesis in nuclei, EdU was labeled with Alexa Fluor555 azide and images were acquired with a Zeiss LSM510 confocal microscope, using a 20×objective (NA = 0.5) and a zoom of 2 as described [45] (link). Pixel intensities were measured for all nuclei, which were demarcated by DAPI staining.