Ta50011 100

Myoblasts were transfected on 96-well plates with full-length mouse PARP-1 cDNA inserted into pCMV6-Entry vector (Myc-DDK-tagged) purchased from Origene Technologies (Cat#MR211449) (Rockville, USA, MD). Insert free plasmid, pCMV-Entry was used as control (Origene, Cat#PS100001). Transfection of myoblasts was performed using Lipofectamine 2000 (Life Technologies), according to the manufacturer's instructions. Briefly, DNA (0.2 μg/well) and Lipofectamine 2000 (1 μl/well) were separately diluted in 25 μl of Opti-MEM (Gibco). Next, DNA was added to the Lipofectamine 2000 reagent and the lipid/DNA mixtures were allowed to form complex for 5 min at room temperature. Cells were washed once with 100 ul of PBS and 100 μl of DMEM containing 15% FBS/well was added to each well. Next, lipid/DNA mixture was added and cells were incubated at 37°C, 5% CO₂. After 24 h, transfection medium was removed and replaced with DMEM containing 2% horse serum to start differentiation. To validate the expression of PARP-1, anti-DDK mouse monoclonal antibody (1:1,000, Origene, Cat#TA50011-100) was used. After 5 days of differentiation, differentiation was confirmed visually (methods as described above) and oxidant sensitivity of the cells was tested by exposing the cells to hydrogen peroxide (0.8 mM) followed by the measurement of LDH release into the culture medium (methods as described above).

Rabbit anti-ODF2 antibody (1:1000 dilution, 12058-1-AP, Proteintech, Rosemont, IL, USA) and mouse anti-α-tubulin antibody (1:1000 dilution, F2168, Sigma-Aldrich, St. Louis, MI, USA) were used for the immunofluorescence assays. Mouse anti-FLAG antibody (1:1000 dilution, TA50011-100, Origene, Rockville, MD, USA) and rabbit anti-HA antibody (1:1000 dilution, 3724S, Cell signaling technology, Danvers, MA, USA) were applied for the Western blotting.

Cells were fixed in ice-cold methanol for 15 min and allowed to dry for 20 min. Rehydrated cells were blocked with 0.25% casein and 0.1% BSA in TRIS-buffered salt solution (TBS) for 30 min. Cells were incubated with rabbit anti-NFAT5 antibody 1:100 (sc-13035, Santa Cruz Biotechnology), ms-anti-DDK 1:1000 (TA50011-100, Origene), or rabbit anti-Ki67 1:100 (ab16667, Abcam) or at 4°C overnight. After washing, cells were incubated with donkey anti-rabbit-Cy3 1:100 (Dianova) for 1 h and mounted with Mowiol (Calbiochem). Nuclei were visualized by counterstaining the cells with DAPI (Invitrogen). Fluorescence intensity was recorded using a fluorescence microscope IX83 (Olympus) and quantitated utilizing the TissueQuest Analysis software version 4.0 (TissueGnostics). In brief, nuclei were visualized by DAPI staining, automatically detected and defined as region of interest (ROI). ROI-associated NFAT5 or DDK fluorescence was automatically detected and displayed as percentage fluorescence-positive nuclei with fluorescence signal above background. In experiments requiring transfection of cells, only successfully transfected cells (as evidenced by detection of DDK in the cytoplasm) were included for further analyses.

The HEK293^MRAP2 cell line was generated by Acrogenic Technologies. In brief, HEK293 cells (ATCC) were cultured in DMEM (high glucose, ATCC) supplemented with 10% FBS and 1% antibiotic-antimycotic (Thermo Fisher Scientific, Gibco) and transfected with Myc-DDK–tagged human MRAP2-expressing plasmid (RC203668, OriGene) using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific, Invitrogen) according to the manufacturer’s instructions. At 48 hours after transfection, cells were cultured in a selection medium containing 500 μg/mL Geneticin (Gibco), which efficiently eliminated 90% of nontransfected parental cells within 72 hours and 100% within 96 hours. The transfected cells were cultured in the selection medium for 4 weeks, and when 90% confluency was reached cells were split at a subcultivation ratio of 1:3. The exogenous expression of DDK-Myc–tagged human MRAP2 in the stable cell pool was evaluated by immunoblotting membrane fractions with mouse anti-DDK (FLAG) monoclonal antibody (1:250; TA50011-100, OriGene) and anti–α-tubulin antibody (CP06, MilliporeSigma) to control for loading. MRAP2 expression was verified with qRT-PCR (forward primer: 5′-ATTTTCTCGCCAAGGCAACG-3′, reverse primer: 5′-TGCTTCTGATGGCTTCCTGG-3′) normalized by β-actin (Supplemental Figure 3). HEK293^MRAP2 cells were maintained in DMEM supplemented with 10% FBS and 1 mg/mL Geneticin at 37°C in 5% CO₂.