Cx 5461

Cells were grown in DMEM (high glucose) supplemented with penicillin and streptomycin, 10% FCS and were cultured at 37 °C and 5% CO₂. U2OS cells were purchased from ATCC and HeLa cells were kindly provided by M. Kühl and were purchased from Sigma (Steinheim, Germany). HEK293A GFP-LC3 cells (ECACC 14050801) were purchased from Sigma (Steinheim, Germany) and were cultured in medium supplemented with 0.4 mg/mL G418 (Sigma, Steinheim, Germany) [34 (link),40 (link)]. Cell lines were regularly tested for absence of mycoplasma and were used in low passage numbers.
HeLa and U2OS cells were seeded in 6-wells and were transfected with 45 nM si RNAs for 48 h using Oligofectamine according to the manufacturer´s instructions. For drug treatment, cells were seeded in 6-wells and grown over night prior administration of DMSO (Sigma) or 1 µM CX-5461 (Caymen, Ann Arbor, MI, USA) for 24 h as indicated. For flux assays in HEK293A GFP-LC3, cells were incubated with DMSO or 1 µM CX-5461 for 20.5 h followed by 50 µM chloroquine (CQ; Sigma, Steinheim, Germany) for 3.5 h.

For RNA Pol I inhibition, cells were treated with 1 μM BMH21 (Selleckchem), 10 ng/ml ActD (Sigma-Aldrich), or 1 μM CX5461 (Sigma-Aldrich) for 45 min or 2 hr. For RNA Pol II inhibition, cells were treated with (1) 5 μg/ml ActD for 2 h, (2) 2.5 μM Flavopiridol (Selleckchem) for 3 hr, or (3) 32 μg/ml DRB for 3 hr. After 3 hr of DRB treatment, cells were washed with PBS for 3 times and were recovered in fresh growth medium for 30 min or 60 min. For epigenetic mark inhibition, cells were treated with 80 nM TSA and 500 nM 5-Aza-dC for 6 days.

NPM1, TCOF1 and p53 anti-rabbit antibodies were obtained from Proteintech (Rosemont, IL, USA). GFP antibody was purchased from Chromotek (Munich, Germany). POL I anti-rabbit antibody and GAPDH anti-mouse antibody were obtained from Santa Cruz (Dallas, TX, USA). The TRPV1 agonist capsaicin was purchased from Sigma-Aldrich (Oakville, ON, Canada). Secondary anti-rabbit and anti-mouse horseradish peroxidase (HRP) conjugated antibodies for Western blotting were obtained from GE Healthcare (Mississauga, ON, Canada). Goat anti-rabbit alexa fluor 555 and 633 conjugated secondary antibodies for immunofluorescence staining were obtained from Molecular Probes (Mississauga, ON, Canada). CX-5461 and CFA were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Anti-Flag (M2, F3165, Sigma), anti-V5 (R960-25, Life Technologies), anti-EXOSC10 (sc-374595, Santa Cruz Biotechnology), anti-EXOSC10 (A303-98A,Bethyl Laboratory), anti-hRrp40 (EXOSC3) (sc-166568, Santa Cruz Biotechnology), anti-EXOSC3 (sc-98776, Santa Cruz Biotechnology), anti-hRrp41(EXOSC4) (sc-166772, Santa Cruz Biotechnology), anti-hRrp4 (EXOSC2) (A303-886A, Bethyl Laboratory), anti-MTR4 (A300-614A, Bethyl Laboratory), anti-Dis3 (A303-765A, Bethyl Laboratory), anti-Nop58 (A302-719A, Bethyl Laboratory), anti-Las1L (A304-438A, Bethyl Laboratory), anti-Puromycin (clone 12D10, MABE343, EMD Millipore), anti-Ub (sc-9133, Santa Cruz Biotechnology), anti-c-Myc (ab32072, abcam), anti-SP1 (07–645, EMD Millipore), anti-Digoxigenin-AP (11093274910, Roche), anti-USP36 (14783–1-AP, Proteintech) and anti-RPL30 (sc-98106, Santa Cruz Biotechnology) antibodies were purchased. Anti-RPL5, anti-RPL11 and anti-RPS27a were described previously (56–58 (link)). Rabbit polyclonal anti-SUMO1 and anti-SUMO2/3 antibodies were provided by Dr Yoshiaki Azuma (University of Kansas). Rabbit anti-USP36 serum was provided by Dr Masayuki Komada (Tokyo Institute of Technology, Japan) (26 (link),29 (link)). Puromycin (Life Technologies), doxycycline (Dox; Sigma), actinomycin D (Act D; Sigma), CX-5461 (Sigma), RNase A (Thermo Scientific) and RNase T1 (Thermo Scientific) were purchased.

Cells were treated with these genotoxic agents at the following doses for 24–72 h prior to downstream analysis with the ALT assays used in this study: pyridostatin/PDS (Sigma) 5 μM; camptothecin/CPT (Sigma) 50 nM; etoposide/ETO (Sigma) 0.5 μM; PJ34 (Calbiochem) 5 μM; veliparib (Stratech) 5 μM; olaparib (Selleckchem) 5 μM; niraparib (Stratech) 5 μM; talazoparib (Selleckchem) 5 μM; CX-5461 (Sigma) 1 μM; hydroxyurea/HU (Sigma) 50–400 μM and aphidicolin/APH (Sigma) 0.2–0.4 μM. Formaldehyde (500 μM, Sigma) was added to cells for one hour, then media was changed and downstream analysis performed at 48–72 h, as with other agents. Negative controls with the drug diluent (water, PBS or DMSO) but without drug were included with each assay.

Chd1^fl/Δ and Chd1^Δ/Δ (established) ES cells were used. Cells were incubated for indicated durations and concentrations with Triptolide (Sigma), Etoposide (Sigma), or CX-5461 (Sigma). Control cells were treated with DMSO. Inhibitors were withdrawn and cells were washed immediately prior to detection of double-stranded DNA breaks and qPCR.