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. 2018 Sep;103(9):e432–e435. doi: 10.3324/haematol.2018.192435

Figure 2.

Figure 2.

Molecular analysis of CCND1 expression in MCL cases. (A) Analysis of CCND1 and CCND2 mRNA expression. RNA was extracted from FFPE material from two lymph nodes (LN1 & 2), two MCL cases positive for CCND1 expression in IHC (MCL-1 and 2) and from the two patients investigated in this study (patients A and B). After reverse transcription, cDNAs were analyzed by quantitative PCR using CCND1 and CCND2 specific primer assays (QIAGEN). GUSB was used as reference gene. (B) Sanger sequencing peaks showing the mutations identified in the two patients investigated in this study. (C) Schematic depiction of the CCND1 gene with the exon/intron structure, the position of the mutations identified in patients A and B and the position of the primers used to analyze the expression of the CCND1a and CCND1b isoformes. The location of the putative binding sites of the antibodies used in this study is also indicated (Ab1: Thermo Fisher Scientific, clone SP4; Ab2: Abcam, EP272Y). (D) Total lysates from HEK 293 cells transfected with plasmids expressing either wild type or mutated CCND1 were resolved on SDS PAGE and analyzed by western blot using two different antibodies against CCND1 (Ab1 and Ab2 as above) and against GAPDH as an internal reference protein. Cells transfected with an empty vector were also analyzed (Vec). (E) PCR analysis of CCND1 isoform expression in FFPE extracted RNA from patients A and B and from 12 CCND1-positive MCL samples. One microliter of cDNA was amplified in a 20 μl reaction containing 4 mM MgCl2, 1 X Q solution (Qiagen), 0.25 mM dNTPs, 0.25 μM each primer, 1.5 Units of AmpliTaq Gold™ DNA Polymerase (ThermoFisher Scientific) and 1 X reaction buffer II with 60 °C as annealing temperature and 45 cycles. For the amplification of total CCND1 we used primers D1EX1_fw and D1EX2_rev2, annealing respectively to exon 1 and exon 2 of the CCND1 gene. For the amplification of the CCND1a isoform we used primers D1EX4_fw2 and D1EX5_rev, annealing respectively to exon 4 and exon 5 of the CCND1 gene. For the amplification of the CCND1b isoform we used primers D1EX3_fw2 and D1EX4_rev, annealing respectively to exon 3 and to the beginning of intron 4 of the CCND1 gene. The sequence of the primers used is provided in Online Supplementary Table S1.