Pcmv6 entry vector system

For luciferase assays, 150,000 cells were transfected with 1.5 μg of plasmid DNA encoding PUM1, PUM2, or an empty plasmid in the pCMV6-entry vector system (OriGene Technologies), plus the full-length or short 3′UTR of SPIN1 or SPIN3 in the psiCheck2 dual luciferase vector system (Promega, Germany) in a 10:1 (plasmid DNA:luciferase vector) ratio. Transfected cells were cultured in 12-well plates for 24 h in standard medium. For PUM transient knockdown experiments, 100,000 cells were transfected with 10 nM siRNA and 150 ng of psiCheck2 vector constructs as described above, and then cultured in 12-well plates for 48 h to achieve effective PUM mRNA depletion. Transfections were performed in three technical repeats per experiment. Cells were lysed and luminescence was measured two times using a Glomax-Multi Detection System luminometer (Promega) and the Dual-Luciferase Reporter Assay (Promega) according to the manufacturer’s protocol. Average Renilla to firefly luciferase luminescence ratios and standard deviations were calculated from three experiments. Luminescence ratios for each combination of constructs and/or siRNA were presented as % relative luciferase units (RLU). The sample transfected with empty pCMV6-entry or control siRNA plus the reporter construct was considered as 100%.

For cell cycle analysis following SPIN or PUM overexpression, 2 × 10⁶ cells were transfected with 30 μg of plasmid DNA encoding SPINs, PUMs, or an empty plasmid in the pCMV6-entry vector system (OriGene Technologies), plus GFP-F in the pEGFP-F vector as a marker of transfected cells (GFP-positive cells) in a 5:1 (plasmid DNA:pEGFP-F vector) ratio. Constructs encoding p21 and p16 or an empty plasmid in the pcDNA3 vector system with GFP-F co-transfection were used as positive controls. Transient knockdown was performed under standard conditions as described above. After transfection, cells were cultured in 15-cm plates for 72 h in standard medium. TCam-2 cells were subsequently trypsinized, washed with PBS, and fixed in cold 100% methanol on ice for 10 min. Cells were incubated at 37°C for 15 min in 50 µg/ml propidium iodide (PI; Sigma Aldrich) containing 330 µg/ml RNAseA (Sigma Aldrich), incubated 1 h on ice, and then analyzed using an S3e™ Cell Sorter (Bio-Rad). Data files were analyzed using ModFit LT software (Verity Software House).

Constructs encoding PUM1 (RC201219), PUM2 (RC211307), SPIN1 (RC201938), or SPIN3 (RC215063), in the pCMV6-entry vector system for protein overexpression were purchased from OriGene Technologies. For luciferase assays, the full-length GAPDH (201 nt) SPIN1 (3429 nt), or SPIN3 (3339 nt) 3′UTRs or their fragments, SPIN1 (195 nt) and SPIN3 (218 nt), were cloned into the psiCheck2 vector (Promega) with the Renilla luciferase ORF, using NotI and XhoI restriction sites.