E4030

To evaluate ITGB1 promoter transcriptional activity, SALL2 KO HEK293 cells were transiently co-transfected with 1 μg of ITGB1 promoter, 0.125 μg of RSV-β-galactosidase (β-Gal), and 2 μg of FLAG_SALL2 (pCDNA.3 FLAG SALL2) or 1 μg of vector control per well. After 24 h, the transfected cells were lysed with reporter lysis 5X buffer (E4030; Promega, Madison, WI, United States) and centrifugated at 14000 × g to pellet cell debris. The supernatant was then assayed for luciferase and β-Gal activity using the manufacturer’s suggested protocols (Promega; E1483 and E4740, respectively). Luminescent reporter activity was measured using a Luminometer (Victor3; Perkin- Elmer). All transfections were normalized to β-Gal activity and performed in triplicate. Luciferase values were expressed as fold induction relative to the pGL3 vector control.

Plasmid containing full-length human MYCBP2 3′-UTR inserted downstream of the luciferase reporter gene was purchased from abmGood (MT-h15016). The luciferase reporter construct, along with β-galactosidase control plasmid, were transfected into 293 T cells. 24 h post transfection, the cells were split into 24-well plates followed by transfection of miR-1247 mimics or all-star negative scrambled mimics at 50 nM. At 72–96 h post miRNA transfection, luciferase activity was determined using a luciferase assay system (E4030, Promega) and normalised to β-galactosidase activity measured with a β-galactosidase enzyme assay system (E2000, Promega). The results represent three independent experiments, each performed in triplicate.

HeLa pTRE-LucIVS2-705 cells were seeded on flat-bottom 96-well plates at 10,000 cells per well in 200 µl complete DMEM. The following day, cells were washed in phosphate buffered saline and topped with premixed 50 µl 2× serum/phenol red-free DMEM (Gibco) + 50 µl peptide cleavage solution (ddH₂O + peptide). Cells were transfected for 30 min at 37 °C with 5% CO₂. The transfection solution was removed, cells were topped with 100 µl complete DMEM. The next day (24-h recovery) cells were washed in PBS. Cells were lysed in 20 µl reporter lysis buffer (Promega E4030) following the manufacturer’s protocol. A volume of 10 μl of lysate from each well was transferred to a 96-well solid black plate; 100 μl of luciferase assay reagent from the Luciferase Assay System (Promega E1500) was added, the plate was agitated for 10 s, and luminescence was measured with a BioTek Synergy 2 plate reader with injector ports. Total protein was measured with a BCA assay (described below). Data were expressed as RLU per µg protein (RLU (µg protein)⁻¹). During screening, positive wells were defined as those whose RLU per µg protein values exceeded the average for the plate by at least three standard deviations. Subsequent PNA705 delivery experiments were conducted as described above except that volumes were scaled for 24-well plate format.

Protein kinase C (PKC) inhibition was assayed in HepaG2 cell lysates using a PKC Kinase Activity Assay Kit (ab139437; Abcam). Cells were grown to 90% confluency in a 60 mm dish. After removal of the medium, 1 mL of lysis buffer (E4030, Promega) was applied for 10 min on ice. Cells were scraped, sonicated, and centrifuged at 15,900 × g/15 min/4 °C (Eppendorf Centrifuge 5415 R; Eppendorf, Stevenage, U.K.). Then, 3 µL of cell lysate was mixed with 297 µL of kinase assay buffer, and 40 µL aliquots were transferred into 0.5 mL microtubes. These aliquots were mixed with 1/100 stock solutions of carvones to obtain final concentrations of 10 µM, 100 µM, and 1000 µM. DMSO (1% V/V) and staurosporine (1 µM) were used as negative and positive controls, respectively. The reaction was initiated by the addition of 10 µL of reconstituted ATP, and the rest of the procedure was performed as described in the manufacturer’s recommendations. Absorbance was measured at 450 nm using an Infinite M200 microplate reader (TECAN, Austria). The results are expressed as a percentage of the negative control. The cell lysate was stored at −80 °C and used in performing three independent experiments.

Cells were washed with ice-cold PBS and lysed with 1X passive lysis buffer (#E4030, Promega) for 15 min with intermittent mixing. The cleared cell lysates were obtained by brief centrifugation and added with firefly luciferase assay substrate (#E1500, Promega) at the ratio of 1:4 (5 µl sample plus 20 µl substrate). The luciferase activity was measured using a Lumat LB 9501 Single Tube Luminometer (Berthold, Hartford, CT). The protein concentration of the cleared cell lysates was also determined using a Bio-Rad DC protein assay kit (Bio-Rad) and used to normalize the luciferase activity, which was expressed as relative luminescence/light unit (rlu).