Cyclin D

Myc, flag, GFP and GST tagged EBNA3C vectors have been described previously [14] (link), [18] (link). pcDNA3-HA-Ub was kindly provided by George Mosialos (Aristotle University of Thessaloniki, Thessaloniki, Greece). Vectors pcDNA3-Cyclin D1, pcDNA3-1x flag-Cyclin D2 and pcDNA3-1x flag-Cyclin D3 were provided by Alan Diehl (University of Pennsylvania School of Medicine, Philadelphia) and used to generate pA3F-Cyclin D by cloning PCR amplified DNA into pA3F vector [4] (link). GST Cyclin D1 vectors were cloned by inserting PCR amplified DNA into pGEX-2TK vector (GE Healthcare Biosciences, Pittsburgh, PA). pGEX-Cyclin D1 (286A) was generated by PCR using pA3F-Cyclin D1 as template. Sh-RNA vector, pGIPZ (Open Biosystems, Inc. Huntsville, AL) and lentiviral packaging vectors were described [38] (link). CDK6 cDNA cloned into pA3F vector was derived from HEK 293 cell RNA that was purified with TRIzol reagent and reverse transcribed with Superscript II (Invitrogen, Inc., Carlsbad, CA). Mouse antibodies to Cyclin D1 (DSC-6) and Sp1 (1C6), and rabbit antibody to Ub (FL-76) were from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Rabbit antibodies to Cyclin D2 and D3 were kindly provided by Alan Diehl (University of Pennsylvania School of Medicine, Philadelphia). Mouse antibodies to flag-epitope (M2) was from Sigma-Aldrich Corp. (St. Louis, MO) and to GAPDH was from US-Biological Corp. (Swampscott, MA). Antibodies to HA-epitope (12CA5) or Myc-epitope (9E10) were prepared from cell culture supernatants as described [14] (link), [18] (link). Mouse (A10) or rabbit antibody to EBNA3C were described [14] (link), [18] (link).
HEK 293, 293T and Saos-2 (p53^-/- pRb^-/-) cells were obtained from Jon Aster (Brigham and Women's Hospital, Boston, MA, USA). Saos-2 and U2OS are human osteosarcoma cell line [39] (link). HEK 293, HEK 293T, U2OS, and Saos-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 50 U/ml penicillin (HyClone, Logan, UT), 50 µg/ml streptomycin (HyClone, Logan, UT) and 2 mM L-glutamine (HyClone, Logan, UT). BL lines BJAB, Ramos, BL41 and B95.8 infected BL41 (BL41/B95.8) were kindly provided by Elliott Kieff (Harvard Medical School, Boston, MA). MutuI, MutuIII were provided by Yan Yuan (School of Dental Medicine, University of Pennsylvania, Philadelphia, PA). These BL lines and LCL1 and LCL2 were maintained in RPMI 1640 (HyClone, Logan, UT) supplemented as described above. EBNA3C expressing BJAB lines were described [14] (link), [18] (link). Unless otherwise stated all cultures were incubated at 37°C in a humidified environment supplemented with 5% CO₂.
Adherent cells were transfected by electroporation with a Bio-Rad Gene Pulser II electroporator as described [14] (link), [18] (link).

Quantitative real time polymerase chain reaction (qRTPCR) was used to study the effect of Tritticum spelta and Salix mucronata plant extract on p53, BCL2, Cyclin D, MMP9 and VEGF gene expression. Total RNAs were extracted from the untreated and the treated HepG2 and Caco-2 cancer cell lines using Gene JET RNA Purification Kit (Thermo Scientific, USA). Then cDNA was synthesized utilizing cDNA Synthesis Kit (Thermo Scientific, USA). Real time PCR was performed using SYBR green master mix and specific primers which are shown in Table 1 in supplementary materials. The 2^−ΔΔCT equation was used to calculate the change in gene expressions in the treated cancer cells relative to untreated cancer cells. The expression of target genes was calculated using the comparative Ct method (threshold cycle number at cross-point between amplification plot and threshold). The CT values of each target gene were normalized to that of β-actin according to manufacturer’s instructions and the change in expression (2^−ΔΔCT) was calculated.

Folin–Ciocalteau reagent, gallic acid (GA), rutin (RU), sulfanilamide, naphthylethylenediamine dihydrochloride, Diphenyl–α-picrylhydrazyl (DPPH), nitroblue tetrazolium (NBT), riboflavin, and vitamin C were supplied from Riedel-de Haën, Germany. DMEM medium and fetal bovine serum (FBS) were obtained from Lonza (USA). Other chemicals were obtained with a high grade from Sigma-Aldrich (St. Louis, MO, USA). Primers for P53, BCL2, Cyclin D, MMP9 and VEGF were purchased from Bioneer, Korea as shown in Table 1 in supplementary materials.