Pcmv6 xl5

Full-length complementary DNAs (cDNAs) of human STIM1 was subcloned into pCMV6-XL5 (Origene)37 (link) with the insertion of enhanced green fluorescent protein (EGFP), YFP or CFP between two additional NarI sites introduced immediately after residue N39. STIM1 mutant constructs were subsequently made using the QuikChange Lightning site-directed mutagenesis kit (Agilent). For STIM1-CFP and STIM1-YFP constructs, human STIM1 was inserted into pECFP-N1 or pEYFP-N1 between XhoI and BamHI. Truncated STIM1-CFP and STIM1-YFP variants were prepared by standard PCR and ligation. pCDNA3.1(+)-mCherry-ORAI1 was made by inserting mCherry between BamHI and EcoRI restriction sites and human ORAI1 between EcoRI and XhoI sites (Life Technologies). NFAT1_1–460-GFP38 (link), mCherry-CAD9 (link), YFP-SOAR10 (link) and YFP-D-SOAR (tandem SOAR domain)39 (link)40 (link) were obtained from Addgene or generated as previously described.

Beta-TC-6 cells were plated on 96-well tissue culture plates and allowed to attach overnight. When 70–80% confluency was reached, cells were transfected with h-proIAPP cDNA clone (vector pCMV6-XL5, Origene, Rockville, MD) or m-proIAPP cDNA clone (vector pCMV6-Kan/NEO, Origene) or h-Hsp72 cDNA clone (vector pEGFP, Addgene, Cambridge, MA) or both h-proIAPP and h-Hsp72 vectors. Transfection complexes were prepared with Opti-MEM (Life Technologies), and Lipofectamine LTX and PLUS reagent (Life Technologies), according to the manufacturer’s instructions. Medium was changed after 4–6 hours. After 48 hours of incubation we used MTS assay to evaluate cell viability.

The human NSCLC lines (NCI-H358, NCI-H1650, NCI-H1568, HCC827, and A549, NCI-H1299) were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). HEK-293 T cell was maintained in DMEM medium (Gibco, USA) and other cells were cultured in RPMI-1640 (Gibco, USA), supplemented with 10% fetal bovine serum (Gibco, USA), and 1% penicillin and streptomycin (Gibco, USA). The cultured cells were maintained at 37°C in a humidified 5% CO₂ incubator. Small interfering RNAs (siRNAs) transfection and plasmid were conducted as described [26 (link)]. When cells reached 40–60% confluence, they were transfected for 48–72 h with siRNAs (circ-in, 5′-GCATCGTGCAGGACTGGAA-3′) targeted to circ_0000677 (constructed by Sangon, Shanghai) at a 50-nM concentration. A scrambled siRNA was utilized as a negative control. Transfection was performed with the lipofectamine 3000 according to the manufacturer’s instructions. To establish stable cell lines overexpressing CCND1, cells were transfected with a pCMV6-CCND1 plasmid DNA (Origene) or the empty plasmid served as the negative control. Human CCND1 cDNA cloned in pCMV6-XL5 was purchased from ORIGENE.

The expression vector pCMV6-XL5 containing the human full-length cDNA for TP53 (TP53-WT) (NCBI Acc.-No: NM_000546.2; SC119832) was obtained from OriGene (Rockville, CA, USA). Originating from TP53-WT, a control vector containing the 637C>T mutation was constructed (TP53-637C>T) by using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies; CA, USA). Briefly, the mutagenic primer hTP53-637C>TFwd 5′-GATGACAGAAACACTTTTTGACATAGTGTGGTGGTG-3′ was used to synthesize a mutant strand by thermal cycling using PfuUltra DNA polymerase followed by DpnI restriction of the methylated parental template. Upon transformation of One Shot TOP10F′ chemically competent E. coli cells and selection, plasmid DNA of ampicillin-resistant colonies was isolated and submitted for sequencing in order to confirm the correct nucleotide sequence (GATC Biotech AG; Cologne, Germany). TP53-WT or TP53-637C>T were transiently transfected using Lipofectamine 2000 transfection reagent (Life Tech; Vienna, Austria) with a DNA (μg) to Lipofectamine (μl) ratio of 1:2.5. Co-transfections with the pEGFP-N1 plasmid (Clontech, GenBank acc. no. U55762; Mountain View, CA, USA) were performed to assure a high percentage of transfection efficiency (>85 %). The appropriate treatment was started 24 h after transfection.