Actinomycin d

RNA stability assay was performed by qRT-PCR as previously reported (76 (link), 81 (link)). Briefly, cells were treated with actinomycin D (10 μg/ml, Fisher) to block transcription, and RNAs were isolated at various time points after actinomycin D treatment. qRT-PCR was then performed using 25 ng of template cDNA for each mRNA gene of interest. Each sample was run in triplicate. The relative abundance of each mRNA was calculated using the ΔΔC_T method and normalized to Gapdh. The relative amount of mRNA at 0 h following actinomycin D treatment was arbitrarily set to 1. Curve fittings of the resultant data were performed using Microsoft Excel and the half-lives of the RNAs calculated.

To evaluate the stability of SIDER2-harboring mRNAs expressed as part of episomal vectors or from the genomic locus, we treated mid-log phase L. infantum promastigotes with 10 µg/mL of actinomycin D (ActD; Gibco-Life technologies) and 2.5 µg/mL sinefungin (Abcam) to arrest de novo transcription and pre-mRNA trans-splicing, respectively. sinefungin was added 5 min prior to ActD (Li et al. 2006 (link); Haile et al. 2008 (link)). Total RNA was isolated at desired time points and analyzed by Northern blotting. To inhibit global protein synthesis, mid-log phase promastigotes were incubated with 10 µg/mL cycloheximide (Sigma-Aldrich), and at various time points, parasites were collected, total RNA isolated and analyzed by Northern blot hybridization. Following transfer, membranes were exposed to a Phosphorimager, and signal intensity was measured using the ImageQuant 5.2 software.

Stability of CFTR mRNA was estimated by administration of Actinomycin-D (5 µg/mL, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), which has long been known to be an inhibitor of RNA-Pol-II-mediated RNA synthesis [29 (link)], and subsequent RT-qPCR-based measurements of CFTR mRNA abundance relative to the time of administration over the following 12 h. ACTB was used as the best control gene for normalization, following comparative optimization of the assay using other controls (18S-rRNA, RPLP0, and CFTR itself at t = 0 h), and considering the higher than average stability of ACTB mRNA [30 (link)]. For the purposes of curve fitting, we assumed exponential mRNA degradation following transcriptional shutdown according to the formula y = (1/B)·e^((A−x)/B), where A and B are position and scale parameters, respectively. Degradation curves were constructed by best fit using the least squares method in the excel add-on solver [31 (link)], and approximate half-lives of CFTR mRNA were estimated.

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Compound C, BAPTA/AM, E64D, pepstatin A and STO-609 were purchased from Calbiochem (San Diego, CA). Actinomycin D was obtained from Gibco® (Grand Island, NY). Iso-tetrandrine was purchased from Wako Pure Chemical Industries, Ltd (Japan). Tetrandrine (Tet) was from Sigma-Aldrich (St. Louis, MO). Fangchinoline was from Chengdu MUST Bio-technology Co. Ltd. (China). Antibodies against p70S6 kinase, phospho-p70S6 kinase (Thr389), AMPKα, phospho-AMPKα (Thr172) were purchased from Cell Signaling Technologies Inc. (Beverly, MA). p62 (SQSTM1) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-LC3 antibody was from Novus Biologicals (Littleton, CO). Anti-β-actin antibody was from Sigma. pEGFP-LC3 reporter plasmid was a gift from Prof. Tamotsu Yoshimori (Osaka University, Japan). Small interfering RNAs and non-targeting control were obtained from Qiagen (Valencia, CA).

Cells were seeded in triplicate in a 6-well plate with 30 × 10⁴ cells per well and allowed to recover for 24 h before drug treatment. Cells were treated with caspase-8 inhibitor (Z-IETD-FMK, FMK007, R&D Systems), caspase-9 inhibitor (Z-LEHD-FMK, FMK008, R&D Systems) or a pan-caspase inhibitor (Z-VAD-FMK, ALX-260-020-M001, Enzo Life Science) at 40 μM, 40 μM or 50 μM, respectively, for 18 h. Cells were treated with either 10 nM vincristine (Cayman Chemical) and incubated for 72 h, 10 nM actinomycin D (Cayman Chemical) for 48 h, 50 μM etoposide (Acros Organics) for 24 h or 0.5 µg/ml TRAIL (PHC1634,Gibco) for 12 h. For multidrug treatment, combinations of etoposide (50 μM) and vincristine (10 nM), or etoposide (50 μM) and actinomycin D (10 nM) were added to the cells and incubated for 24 h. Cell viability was determined using trypan blue staining, and cell number was counted in three different wells on blinded samples. For DNA methyltransferase inhibition assay, RH30 cells were treated with either DMSO (vehicle) or 5-aza-2′deoxycytidine (Sigma-Aldrich) at 30 µM and 60 µM for 48 h prior to RNA isolation. Culture medium supplemented with fresh drug was changed every 24 h. All assays were performed at least twice to confirm results.

NHEK were co-cultured in 6 or 12 well plates with γ-irradiated 3T3 murine fibroblasts as feeder to support keratinocyte growth in different RM⁺ formulations for 3 days before removing the feeder and using keratinocytes for gene expression analysis by qPCR (cells grown in 12 well plates) and western blotting (cells grown in 6 well plates). For specific treatment, the co-cultures were seeded in 12 well plate, grown in RM⁺ containing CS-FCS, next day the cells were treated with reagents, ATRA (R2625, Sigma) or ED (E8875, Sigma) for 24 h. ATRA was first dissolved in DMSO and further diluted in ethanol (EtOH) to the desired concentrations. The concentrations of DMSO/EtOH used as a vehicle control varied from 0.003% to 0.03% (v/v). ED was dissolved in DMSO and further diluted in culture medium to working dilutions. DMSO (0.001%) was used as a vehicle control. PR (P3532, Sigma, UK) was added in PR free RM⁺ culture medium at 0.01 mg/ml. The feeders were removed by squirting the culture with PBS/EDTA and collecting cell lysates for expression analysis by qPCR. When PR was added, PR free medium was used to grow cells as control. Actinomycin D (AD, 11805-017, Gibco, UK) was diluted from a stock (2 mg/ml in water) to working dilutions.