Cignal myc reporter assay kit

The eGFP Myc transcriptional activity was assayed as previously described [26 (link)]. Briefly, cell transfection was performed using TransIT-2020 transfection reagents according to the manufacturer’s instructions. LNCaP cells were plated at 10,000 cells per well and treated for 1 day with the indicated concentration of compound with increasing concentrations (0−25 μM) of compounds for screening and measuring IC₅₀. Myc reporter activity was measured using the Cignal Myc Reporter Assay Kit from Qiagen (#336841; Hilden, Germany) according to the manufacturer’s instructions.

A total of 30,000 cells in 24-well plates were used. The experiment was performed using the Cignal Myc Reporter Assay Kit (Qiagen, Hilden, Germany), according the manufacturer’s instructions.

c-Myc activity was determined using the cignal Myc reporter assay kit (CCS-012L) from Qiagen (Germantown, MD, USA). Myc reporter assay is made to keep track of the Myc signaling pathway’s activity in cells. A transfection-ready expression vector for c-Myc and a reporter vector for luciferase are included in the kit. As a first step, HEK293 cells were transfected with a c-Myc-luciferase reporter construct and cultured according to the manufacturer’s suggestions. HEK293 cells were used as hematopoietic cells are difficult to transfect. Cells were then treated with different concentrations of cynaropicrin or DMSO (negative control), or the known Myc inhibitor (10058-F4) (positive control) for 48 h. The activity of the c-Myc promoter was quantified using the Dual-glo^® Luciferase Reporter Assay System (E2920, Promega, Madison, WI, USA). An Infinite M2000 Pro™ plate reader (Tecan, Germany) was used to measure the luminescence of firefly and renilla luciferases [47 (link)]. $\mathrm{c}-\mathrm{Myc}\mathrm{activity}=\left(\mathrm{firefly}\mathrm{luciferase}\mathrm{luminescence}/\mathrm{renilla}\mathrm{luciferase}\mathrm{luminescence}\right)$ $\mathrm{Relative}\mathrm{luciferase}=100\times \left(\mathrm{firefly}\mathrm{luciferase}\mathrm{luminescence}/\mathrm{renilla}\mathrm{luciferase}\mathrm{luminescence}\right)$ $\mathrm{Normalized}\mathrm{c}-\mathrm{Myc}\mathrm{activity}=\mathrm{relative}{\mathrm{luciferase}}_{\left(\mathrm{sample}\right)}/\mathrm{relative}{\mathrm{luciferase}}_{\left(\mathrm{DMSO}\right)}$

Luciferase assay was performed with modified protocol previously described [53 (link)]. Briefly, DLD1 and HCT116 cells were transiently cotransfected with the Myc/Max firefly luciferase construct (100 ng) and the Renilla luciferase vector (10 ng) to normalize transfection efficiency (Cignal Myc Reporter assay kit, QIAGEN, Hilden, Germany). After 18 h from transfection, a dual luciferase assay was performed according to manufacturer’s instruction (Promega, Madison, WI, USA). Luciferase readings were measured using an EnSpire-2300 luminometer (Perkin Elmer, Waltham, MA, USA). Data were represented as relative luciferase activity, obtained by the ratio of firefly values (promoter reporter) to Renilla values (control reporter). Experiments were repeated at least three times, and values are expressed as the mean ± SD.

LNCaP-NMYC cells were transfected using TransIT-2020 transfection reagents per the manufacturer’s protocol. Cells were plated at a density of 10,000 cells per well of a 96-well plate. Following 24 h treatment with 5 µM or 10 µM of the compounds, the Myc reporter activity was measured using the Cignal Myc Reporter Assay Kit from Qiagen (#336841) (Hilden, Germany) per the manufacturer’s instructions.

The Qiagen cignal MYC reporter assay kit (CCS-012L, Germantown, MD, USA) was used to examine the impact of adapalene on c-MYC activity, as previously described [29 (link)]. Briefly stated, HEK293 human embryonic kidney cells were transfected with a c-MYC-luciferase reporter construct and grown in accordance with the instructions provided by the manufacturer. Cells were then exposed to adapalene (5 and 10 µM), DMSO as negative control, or 10058-F4 (127.5 2 µM) as positive control. After 24 h, the Dual-glo^® Luciferase Reporter Assay System (E2920, Promega, Madison, WI, USA) was added to each well to quantify the activity of the c-MYC promoter by measuring the luminescence of firefly and renilla luciferases using an Infinite M2000 Pro^TM plate reader (Tecan, Crailsheim, Germany).
$$\mathrm{c}-\mathrm{M}\mathrm{y}\mathrm{c} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\mathrm{f}\mathrm{i}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{l}\mathrm{y} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}}{\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{i}\mathrm{l}\mathrm{l}\mathrm{a} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}}$$
$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e}=100\times \left(\frac{\mathrm{f}\mathrm{i}\mathrm{r}\mathrm{e}\mathrm{f}\mathrm{l}\mathrm{y} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}}{\mathrm{r}\mathrm{e}\mathrm{n}\mathrm{i}\mathrm{l}\mathrm{l}\mathrm{a} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{e}}\right)$$
$$\mathrm{N}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{z}\mathrm{e}\mathrm{d} \mathrm{c}-\mathrm{M}\mathrm{y}\mathrm{c} \mathrm{a}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}{\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{l}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{e} \left(\mathrm{D}\mathrm{M}\mathrm{S}\mathrm{O}\right)}$$

Cells (30,000) were analysed per experiment in 24-well plate using the CignalMyc Reporter Assay Kit (Qiagen, Hilden, Germany), according the manufacturer’s instructions. Because c-MYC expression is dependent on cell density, all the experiments were performed with cells at 70–80% confluency in serum-deprived medium.