Lamin b1

HeLa cells were grown on minimal essential medium containing 10% newborn calf serum. HSV-1 wild type strain KOS, null mutant 27-LacZ, N-YFP-tagged ICP27 (N-YFP-ICP27) and n504 were previously described31 (link). Cells were infected with wild-type or mutant virus as indicated for 8 hours at a multiplicity of infection (MOI) of 10 for single infections and a MOI of 5 for co-infections and incubated at 37 °C. Transfection of plasmid DNA was performed by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Transfected cells were infected 24 hours after transfection with 27-LacZ to stimulate expression of the native ICP27 promoter by the virion tegument protein VP16 as previously described58 (link). Eight hours after infection, cells were harvested, and immunoprecipitation was performed on cell lysates using GFP/YFP antibody Ab290 (Abcam) as described previously6 (link). Immunoprecipitated complexes were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Western blot analysis was performed as described previously6 (link) with anti-ICP27 antibodies P1119 and P1113 (Virusys), anti-GFP/YFP (Ab290; Abcam), anti-GFP antibody Ab1218 (Abcam) and Lamin B1 (Abcam).

Proteins were extracted using RIPA buffer with 1% Protease inhibitor cocktail (Nacalai Tesque). After determination of the protein concentration using Protein Quantification Assay (Takara Bio Inc.), all samples were denatured in Laemmli sample buffer for 5 min at 100 °C. The denatured samples were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% milk or 5% BSA, the membranes were incubated with the primary antibodies as follows: p16^INK4a (1:1000, IBL, 11104), p21^Cip1/Waf1 (1:1000, Cell Signaling Technology, 2947), Lamin B1 (1:5000, Abcam, ab16048), RB (1:500, Santa Cruz, sc-102), phospho-RB (Ser780) (1:1000, Cell Signaling Technology, 9307), p53 (1:1000, Santa Cruz, sc-6243), phospho-p53 (Ser15) (1:1000, Cell Signaling Technology, 9284) and α-Tubulin (1:2000, Sigma-Aldrich, T9026). The membranes were then incubated with the secondary antibodies as follows: anti-mouse IgG (1:2000, Cell Signaling Technology, 7076), anti-rabbit IgG (1:2000, Cell Signaling Technology, 7074) and visualized with Amersham ECL prime/select (GE Healthcare), followed by detection with chemiluminescence using LAS-3000mini imaging system (Fujifilm) and by analysis of data using Multi Guage V3.1 (Fujifilm). Uncropped and unprocessed scans of the blots are included in the Source data file.

Whole-cell and nuclear protein (Minute™ Cytoplasmic & Nuclear Extraction Kits; Invent Biotechnologies, Eden Prairie, MN, USA) was separated using 7.5% or 10.0% SDS-PAGE, transferred onto nitrocellulose membranes, and probed with antibodies against Nrf2 (1:15,000; Abcam), p-Nrf2 (Ser40) (1:15,000; Abcam), Keap1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated p62 (phospho-p62, Ser351, 1:1000; Medical & Biological Laboratories Co., LTD, Woburn, MA, USA), STAT3 (1:1000; Cell Signaling Technology), phosphorylated STAT3 (phospho-STAT3, Thy705, 1:500; Cell Signaling Technology), Lamin B1 (1:10,000; Abcam), and β-actin (1:10,000; Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation at 4 °C, the membranes were washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and visualized using an ECL prime detection kit (GE Healthcare, Buckinghamshire, UK).

DMEM low glucose (Cat No E15-806), DMEM-F12K (Cat No E15-012), foetal calf serum (FCS)(Cat No A15-104) and trypsin (Cat No L11-659) were purchased from PAA Laboratories, UK.
Antibodies to Programmed Cell Death Gene 4 (PDCD4) (Cat No ab51495), β-actin (Cat No ab8226), Lamin B1 (Cat No ab8226), HIF1-alpha (Cat No ab51608) and FITC conjugated anti-rabbit secondary antibodies (Cat No ab6717) were purchased from AbCam, UK. HRP-conjugated anti-rabbit (Cat No A0545), anti-mouse secondary antibodies (Cat No A5278), goat serum (Cat No G9023) and all laboratory chemicals and reagents were purchased from Sigma, UK unless otherwise stated.

Immunoprecipitation experiments were performed as previously described (25 (link)). Lysates were prepared from 2.5 × 10⁶ cells using RIPA buffer (0.1% SDS, 0.5% Na-deoxycholate, 1% NP40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris/Cl, pH 8) supplemented with phosphatase, protease inhibitors and benzonase. One milligram of lysate was incubated overnight at 4°C with BcMag^TM Magnetic Beads (Bioclone) conjugated with 4 μg of anti-RECQ1 antibody under rotation, according to the manufacturer instructions. After extensive washing in RIPA buffer, proteins were eluted in 2× electrophoresis buffer and subjected to SDS–PAGE and western blotting.
Western blotting were performed using standard methods. Blots were incubated with primary antibodies against RECQ1 (Santa Cruz Biotechnology), SMARCAL1 (Bethyl), MRE11 (Novus Biological), DNA2 (Abcam), EXO1 (Santa Cruz Biotechnology), anti-PAR (Abcam), tubulin (Sigma-Aldrich) and lamin B1 (Abcam). After incubations with horseradish peroxidase-linked secondary antibodies (Jackson Immunosciences), the blots were developed using the chemiluminescence detection kit ECL-Plus (Amersham) according to the manufacturer's instructions. Quantification was performed on scanned images of blots using the Image Lab software, and the values shown on the graphs represent normalization of the protein content evaluated through lamin B1 or tubulin immunoblotting.