Dactinomycin

RNA extraction, cDNA synthesis and quantitative PCR were processed as described in Sagredo et al. [33 (link)]. ACTB was used as a housekeeping gene. In addition, RESS-qPCR assays for AZIN1 and MDM2 targets were performed according to Crews et al. [34 (link)]. The complete primer list is presented in Additional file 6. mRNA stability of ATM, GINS4 and POLH, in ZR-75-1 SHC and SHADAR cells were assessed using Dactinomycin (SIGMA-Aldrich) (3 µg/mL) at 0, 6, 8 and 16 h, and total RNA was extracted to analyze the different gene of interest using qRT-PCR. To compare the mRNA decay curves, each condition was normalized against the respective group of 0 h treated cells.

For the transcription inhibition with D-Actinomycin, RKO cells transfected with a scrambled or IMP3 specific shRNA were seeded in a density of 2.5 x 10⁵ in 6-well-plates. The next day, medium was exchanged to fresh DMEM containing either 5 µg/mL D-Actinomycin (Sigma Aldrich, Israel) or equal volume DMSO as diluent control. D-Actinomycin effectively inhibits the elongation process of the RNA polymerase II, thereby suppressing the synthesis of new mRNA transcripts (Sobell, 1985 (link)). After 16 hr incubation, medium was removed, and cells were harvested following the manufacturers’ protocol for RNA extraction (Zymo Research, CA, USA). cDNA was prepared using M-MLV reverse transcriptase (Invitrogen, CA, USA) according to manufacturer’s protocol. qRT-PCR was performed as mentioned in the corresponding section. For analysis, we defined the level of a specific mRNA in the DMSO-treated control cells (diluent control) in both the scrambled shRNA- and shIMP3-transduced cells as 1. By calculating the ratio of mRNA level after 16 hr D-Actinomycin treatment and 16 hr DMSO treatment, we assessed the rate of mRNA decay in dependence of IMP3.

The Pd(II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was obtained from Porphyrin Products (Logan, UT, USA). Dactinomycin (actinomycin D, MW ≈ 1,255) was purchased from Merck (Whitehouse Station, NJ, USA). A lyophilized powder of caspase inhibitor I (zVAD-fmk, MW ≈ 467.5) was purchased from Calbiochem (La Jolla, CA, USA). Ac-DEVD-AMC (MW ≈ 729.6) and caspase-3 (molecular mass ≈ 30.5 kDa, heterodimer active human recombinant) were purchased from Axxora LLC (San Diego, CA, USA). Glucose [anhydrous], bovine serum albumin (free of endotoxin and fatty acids), and remaining reagents were bought from Sigma-Aldrich (St. Louis, MO, USA).
Dactinomycin solution was made fresh in dH₂O; its concentration was determined by absorbance at 440 nm, using an extinction coefficient of 24,450 M⁻¹ cm⁻¹ [31 (link)]. The zVAD-fmk solution (2.14 mM) was made by dissolving 1.0 mg of zVAD-fmk in 1.0 ml of dimethyl sulfoxide and stored at −20°C. The Ac-DEVD-AMC caspase substrate was dissolved in dimethyl sulfoxide at a concentration of 6.85 mM and stored at −20°C in small aliquots. Phosphate-buffered saline (PBS) with glucose (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, and 5 mM glucose; pH 7.4) was made fresh. NaCN solution (1.0 M) was prepared in dH₂O; the pH was adjusted to ~7.0 with 12 N HCl and stored at −20°C.

A custom‐arrayed anticancer library was used (Bezu et al, 2018). In addition, bortezomib (5043140001); cisplatin (C2210000); crizotinib (PZ0191); dactinomycin (A1410); daunorubicin (D0125000); docetaxel (01885); doxorubicin (D1515); epirubicin (E9406); flavopiridol (F30055); ISRIB (SML0843); β‐lapachone (L2037); mitoxantrone (M6545); mycophenolate mofetil (SML0284); nonoxynol‐9 (542334); paclitaxel (T7191); thapsigargin (T9033); tunicamycin (T7765); vinblastine sulfate (V1377); and vincristine sulfate (V0400000) have been bought from Sigma‐Aldrich. Dactolisib (BEZ235) (sc‐364429) came from Santa Cruz biotechnology (Dallas, TX, USA). Oxaliplatin came from Accord Healthcare (Ahmedabad, India). Topotecan (609699), 7‐hydroxystausporine (UCN‐01) (72271), becatecarin (101524), 5‐fluorodeoxycytidine (B86) (515328), and RH‐1 (394347) were kindly provided by the National Cancer Institute (NCI). Lurbinectedin (PM01183) came from PharmaMar (Madrid, Spain).

Vincristine (VCR, #S2141), afatinib (#S1011), tariquidar (#S8028), lapatinib (#S2111), ceritinib (#S7083), copanlisib (#S2802), and decitabine (#S1200) were purchased from Selleckchem (Houston, TX, USA). Verapamil (#V4629), dactinomycin (#A4262), and hydroxychloroquine (#C6628) were purchased from Sigma–Aldrich (Merck KGaA, Darmstadt, Germany). BKM120 (#11587) and panobinostat (#13280) were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Dasatinib (#D‐3307), everolimus (#E‐4040), and olaparib (#O‐9201) were purchased from LC Laboratories (Woburn, MA, USA). Staurosporine (STS, #HY‐15141) and larotrectinib (#HY‐12866) were purchased from Medchem Express (Monmouth Junction, NJ, USA). Bortezomib (#CT‐BZ001), trametinib (#CT‐GSK112), and venetoclax (#CT‐A199‐2) were purchased from ChemieTek (Indianapolis, IN, USA). The ERBB4 blocking antibody (#MA513016) was purchased from Invitrogen (Thermo Fisher Scientific Inc.).