## 3 Results and Discussion
### Proofs of Concepts: Western Blotting Analysis  
With the aim to verify the correctness of the NMF results, the expression of some proteins codified by the topmost twenty genes that are part of the identified metagene were found in six representative HMCLs (U266, SKMM2, KMS12 BM, NCIH929, RPMI8226, MM1S) by using the western blotting technique [44; 45; 46; 47]. One or two HMCLs were selected from each cluster, based on their different responses to the clinically used drugs. In Figure 5, the expression of some specific proteins, analyzed by western blotting analysis and involved in the eicosanoid biosynthesis related to the arachidonic acid pathway of various human myeloma cell lines, is depicted. The expression extent values of each protein were assigned as listed in Figure 8.  
U266-B1 is a B lymphocyte plasmacytoma cell line belonging to cluster 1 characterized by the expression of MG1-2-3-5 (depicted from the colored boxes under cell line name, Figure 8). _HSD17B4_, the gene coding for the protein HSD417B, was expressed at 8% in MG1, at 3% in MG3, at 5% in MG4, and 4% in MG5 (gene percentages are indicated in the colored boxes, Figures 7 and 8) but its expression (0.37) in U266-B1 was due only to MG1, 3, and 5. In fact, U266-B1 (cluster 1, Figure 5) was not represented by MG4 and HSD17B4 was not present in the top most 20 genes of MG2. In U266-B1, COX-1 was not detected by western blot analysis (Figure 6) thus it was supposed that PTGS1 is downregulated in this cell line. GGT1 expressed at 8% in MG2 codifies the corresponding protein which was found with a value of 0.18 in U266-B1. Comparable expression values 0.17 and 0.26, respectively, were found for LTA4H and ALOX15B; LTA4H was represented by all 5 MG with a preponderant contribution of MG1 in which LTA4H was expressed at 7% of all genes. ALOX15P was found in MG1, 3, 4, and 5 but its expression was higher in MG3 (7%). Among the analyzed proteins, PGES2 and ACAA1 were highly expressed in U266-B1 with a value of 19.9 and 2.17, respectively. PGES2 was found in MG1, 2, and 5 with no involvement of MG3 and 4 which do not compose the U266-B1 cell line. ACAA1 derive from MG1, 3, and 5, with no contribution of MG 2 and 4. CBR1 and CYP1A2 were not detected in U266-B1, even though their corresponding genes were represented in MGs 1-5. Their absence could be justified by the downregulation of CBR1 and CYP1A2. SK-MM-2 and KMS-12-BM, both belonging to cluster 2 (Figure 5), had a different metagene composition as depicted by the colored boxes under cell line name (Figure 8). Figure 8: Protein expression in all studied cell lines (Expression values are reported with respect to HEK293 wild type or HepG2 cell line were used as a positive control, as suggested by the antibody data sheet, to which the value 1 has been arbitrarily assigned. Color codes are the same used for metagenes 1–5 depicted in Appendix A. All data are related to HEK293-COX-1, the value = 1 was assigned to the expression of COX-1. NF = not found). SK-MM-2, a B lymphocyte plasmacytoma cell line, was mostly composed of metagene 3 and 1, with a little contribute given by metagene 2. From western blotting analysis (Figure 9) of this cell line, it was evident that all the proteins codified by the genes of metagenes 2 and 3 were expressed, except for CYP1A2. Concerning KMS-12-BM, a multiple myeloma cell line, it had an almost similar contribution from all metagenes with the exception of metagene 5. In this case, PGES2 was the major expressed protein while for COX-1, CBR1, and CYP1A2, no expression was detected (Figure 9). NCI-H929, another multiple myeloma cell line, belongs to cluster 3 with a marked expression of genes of metagene 4, and in part of metagenes 1, 3, and 5. Also, in this cell line, the expression of PGES2 was high (439) with respect to the positive control. CBR1 and CYP1A2 were not found in U266-B1, while a good expression of ALOX15B, HSD417B, ACCA1, and COX-1 was observed.