Ps100010

Cells were transfected with a plasmid containing turbo green fluorescence protein (GFP) (Origene (Rockville, MD, USA), PS100010) or corresponding plasmid containing C-terminally tagged ANXA6 transcript variant 1 (Origene, RG202086) using Opti-MEM® (Life Technologies, 11058-021) and Lipofectamine LTX reagent (Life Technologies, 15338-100) (DNA:LTX ratio was 1:4).

Equal amount of GFP (OriGene, PS100010), OTUB1‐GFP (OriGene, MG203606) or CCN6‐FLAG (OriGene, MR223941) + K48 Ub‐HA (Addgene, #17605) plasmids were transfected into 4T1 cells with Lipofectamine 3000 (1 μg plasmid/1 million cells). Except for the K48 residue, all other lysines were mutated to arginines in the K48 Ub‐HA plasmid. Similarly, in the K63 Ub‐HA plasmid, all lysines except for K63 were mutated to arginines. After 24 h, GFP, OTUB1‐GFP and K48 ubiquitinated CCN6 were purified from cell lysates by immunoprecipitation. Immunoprecipitated samples were washed sequentially with PBS and deubiquitination buffer (5 mM MgCl₂, 5% glycerol, 50 mM Tris‐HCl, 2 mM ATP‐Na₂ and 2 mM DTT). Then, K48 ubiquitinated CCN6 was incubated with GFP or OTUB1‐GFP in the deubiquitination buffer at 37°C for 2 h. Thereafter, samples were analysed by Western blot.

The cells were treated with siRNA 24 h prior to DNA plasmid transfection. Appropriate CMV6-AC-RNase H1-GFP (Origene-PS100010) (2.5 μg per 6 cm dish and 1 µg per well of a 6 well plate) was mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM, incubated for 30 min and then added to cells at 70% confluency growing in DMEM supplemented with 8%FCS. After overnight incubation at 37 °C, the Opti-MEM transfection mixture was removed and replaced with DMEM medium supplemented with 8% FCS and incubated for a further 48 h.
For the CNOT1 complementation experiments, MCF7TRCNOT1 KD cells, grown to 70% confluency, were treated with doxycycline for 48 h and then transfected with a plasmid expressing Flag-tagged CNOT1 (pcDNA3-CNOT1, GenScript, Piscataway, NJ, USA, clone ID: OHu08018) in a similar manner to CMV6-AC-RNase H1-GFP. The CNOT1 plasmid was not siRNA-resistant but the addition of large amounts of exogenous DNA led to an appreciable increase in the levels of CNOT1 protein, as shown in Figure 1H.

To establish stable cell lines expressing SNCG, SUM cells were transfected with a pCMV6-SNCG-GFP plasmid DNA (Origene, RG204173) or with the corresponding empty vector pCMV6-A-GFP plasmid DNA (Origene, PS100010) as a control. Briefly, one day before transfection, 2×10⁵ SUM cells/well were plated in a 6-well plate. Cells were transfected with 1.5 μg/well of the recombinant plasmid or the empty plasmid as control, respectively, using jetPRIME^® (Polyplus) according to the manufacturer's transfection protocol. Fresh growth medium was replaced after 24 h of transfection. Between 5 and 7 days after transfection, GFP-positive SUM cells were successively sorted three times by using a FACSAria II cell sorter (BD Biosciences) to a purity of >85%, and analyzed.
For siRNA experiments, T47D and MCF7 cells were transfected with SNCG siRNA and control siScramble (siSrc) constructs (Origene, SR304497) and SUM-SNCG-GFP and SUM-CTL-GFP cells were transfected with p21 siRNA and control siScr constructs (Origene SR300740), using jetPRIME^® (Polyplus) according to the manufacturer's transfection protocol. Total RNA and proteins were extracted 48 h after transfection for analysis. For irradiation experiments, cells were irradiated 48 hours after transfection.

Plasmids, comprising 91 (91S) or 70 (70S) ‘GGGGCC’ hexanucleotide repeats under control of a T7 promoter were obtained and adapted as described earlier [46 (link), 64 (link)]. In the 70S plasmid, a reverse complementary sequence of the T3 promoter was inserted 30 bp after the repeats to generate antisense transcripts (70AS). GFP control plasmid construct (PS100010, Origene, Rockville, USA) was digested with AgeI (ER1461, Thermo Scientific, Waltham, US) restriction enzyme. FLAG-tagged HNRNPK (RC201843, Origene) and RRM2 (RC228362, Origene) plasmid constructs were digested with AgeI.
HNRNPK deletion constructs were synthesized by Genscript (Piscataway, USA) in a pUC57 vector and subcloned in a pCMV6-Entry vector (PS100001, Origene). After XhoI and EcoRI restriction digestion of donor constructs, desired inserts were harvested using the GenElute Gel Extraction kit (Sigma, Saint-Louis, US), according to the manufacturer’s instructions. Next, inserts were ligated (16 °C, overnight) with T4 ligase into the pCMV6 vector. These constructs were then transformed in E. coli TOP10 chemically competent cells (C404010, Invitrogen, Waltham, US). Consequently, plasmids were linearized with AgeI restriction enzyme.