Fig. 1.

Elevated AHSA1 expression confers poor survival of MM patients and promotes MM cell proliferation. A HuProt™20K Proteome Microarray Chip indicated the top 5 protein targets binding to Bufalin. The yellow arrow indicated positive protein interacted with Bufalin, and the blue arrow represented the negative control. B Among the top 5 proteins, AHSA1 was the exclusive gene. The signal level of AHSA1 was shown on the y-axis. Patients designated as healthy donors with normal bone marrow plasma cells (NP, n=22), monoclonal gammopathy of undetermined significance (MGUS, n=44) or multiple myeloma (MM, n = 351) were sorted on the x-axis. C-D Increased AHSA1 mRNA expression was positively associated with poor overall survival (OS) in first diagnosis and relapsed MM patients from (C) TT2 and (D) HOVON65 patient cohort. Events/N means events of death/total patients. E Representative Immunohistochemistry staining on primary MM samples (n=14) and normal controls (n = 5). F Microscale thermophoresis (MST) analysis for the interaction of Bufalin with human AHSA1 recombination protein. G Validation of AHSA1 overexpression in AHSA1-OE ARP1 and H929 cells relative to control cells. H Cell cycle analysis for WT and AHSA1-OE cells. I Representative images of cell colonies of WT and AHSA1-OE cells in soft agar. J Confirmation of AHSA1 protein knockdown in ARP1 and H929 cells after transfection with AHSA1 shRNA. K Representative images of cell colonies of WT and AHSA1-KD cells in soft agar. L Cell cycle analysis for WT and AHSA1-KD cells. M WB analysis of PARP and Caspase 3. The data are expressed as mean ± SD.*p<0.05, **p<0.01, ***p<0.001