Prl cmv

A DNA sequence encoding the promoter region of mouse Rpl3 (from the transcription start site to 1.2 kbp upstream) was cloned into the pGL2-basic vector (Promega), and the resulting construct together with pRL CMV (Promega) was introduced into C2C12 cells with the use of the FuGENE HD Transfection Reagent (Promega). After transfection for 2 days, luciferase activities were assayed with the use of a Dual-Luciferase Reporter Assay System (Promega) and Lumat LB 9510 Tube Luminometer (Berthold Technologies).

FLAG-tagged full length HDAC9 (HDAC9FL) plasmid was a kind gift from Dr A. Zelent [15 ]. For stable transfections, pcDNA3.1-HDAC9-puromycin plasmid was obtained by subcloning HDAC9FL into the pcDNA3.1 vector (Invitrogen, Cergy Pontoise, France) conferring puromycin resistance. The PWWP reporter construct containing the promoter of the CDKN1A gene was previously described [17 (link)]. The pRL-CMV (Promega, Charbonnières, France) was used for normalization. Zeocin was purchased from InvivoGen (Toulouse, France). HDAC inhibitors TSA and LBH 589 were purchased from Sigma Aldrich and Alinda Chemicals Ltd respectively.

ATX-3^+/+ and ATX-3^-/- MEF cells were co-transfected with the indicated reporter constructs PG13 and the internal control Renilla luciferase pRL-null (pRL-CMV, Promega) at a ratio of 8:1 using PEI. Luciferase assays were performed using a dual-luciferase reporter assay system (E1910, Promega) according to the instructions of the manufacturer. Data were normalized for activity of Renilla luciferase to account for transfection efficiency. The assays were performed in duplicate, and data represent the average of five independent experiments.

H1299 cells were transfected with pRL-CMV (Promega), pBV-Luc BDS-2 3x WT (pBDS-2) (BDS-2 3x WT (p53 binding site) was a gift from Bert Vogelstein (Addgene plasmid #16515; http://n2t.net/addgene:16515; RRID:Addgene_16515; [56 (link)])), and either pcDNA3.1(+) as an empty vector control, pcDNA3.Myc-p53 alone or pcDNA3.Myc-p53 in combination with increasing amount pcDNA3.Myc plasmids encoding the indicated p63 variants. The total amount of transfected plasmid DNA was kept constant by addition of pcDNA3.1(+) if necessary. Apart from that, the luciferase reporter displacement assay was performed as the conventional variant described above.
For all luciferase reporter assays presented in this study at least three independent biological replicates were performed. Statistical significance was assessed by ordinary one-way ANOVA followed by Tukey’s post hoc test using Graphpad Prism 8 (Supplementary Table 3).

For luciferase assays, lipofectamine 2000 (Invitrogen) was used to transfect cells with the pGL3 promoter vector containing the Cryba1 promoter spanning 100 bp on either side of the STAT3 binding site located 2750 bp upstream of the promoter. The vector containing Cryba1 promoter is mentioned as pGL3-Cryba1 promoter. We also cloned 100 bp on either side of the STAT3 binding site at 1942 bp in intron 2; this vector is referred to as pGL3-Cryba1 intron. To generate the Cryba1 promoter deletion construct, the region containing −2774 to −2756 bp was deleted from the pGL3-Cryba1 promoter vector using In-Fusion^R HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA) following the manufacturer's instructions. This vector is referred to as pGL3-Cryba1ΔSTAT3. The Notch luciferase reporter construct (2.3 µg) together with pRL-CMV (0.1 µg; Promega, Madison, WI) carrying the Renilla luciferase gene under the control of the cytomegalovirus promoter was co- transfected. Luciferase activity was measured using a luciferase assay system (Promega). The reporter activity was calculated by normalizing the firefly luciferase value with that of the Renilla luciferase control vector and expressed as relative luciferase units. All data were obtained from at least three independent experiments. The relative promoter activities were depicted as the mean ± S.D.