Figure 3.

HMGB1 is overexpressed in MM and contributes to MM cell survival. (A) The expression of HMGB1 from microarray analysis of samples obtained from healthy donors and from MGUS, SMM and MM patients. Data presented as the mean±SD. (B) Kaplan-Meier analyses of overall survival (OS). Publicly available microarray data sets GSE 5900 and GSE 2658 were downloaded and data were reproduced to estimate the OS. (C) Expression of HMGB1 mRNA in primary human BM CD138⁺ cell. Primary human CD138⁺ cells were isolated from the BM aspirates of normal subjects (n=9) and patient with myeloma (n=19). GAPDH normalized HMGB1 mRNA level was analyzed by quantitative RT-PCR. (D) HMGB1 expression in myeloma cell lines and B-cell was measured by Western blotting. GAPDH was used as a loading control. Densitometry analysis of HMGB1 intensity was performed using ImageJ software, normalized with loading control (HMGB1/GAPDH) and plotted on a bar diagram. (E) A CCK-8 assay was used to check cell viability after ARP-1 cells were transiently transfected with HMGB1 shRNA or scramble shRNA for various times. Data are presented as the mean±SD (n=3, **p<0.01). (F) The growth curve of ARP-1 cells transfected with shHMGB1. ARP-1 cells were transiently transfected with scramble or HMGB1 specific shRNA and cultured with a density of 1×10⁵ cells/ml. Total number of Trypan Blue negative cells were manually counted at the indicated time points and plotted (**p<0.01). (G) ARP-1 cells were transiently transfected with a vector encoding HMGB1 (ovHMGB1) or a blank (Mock) vector for different time and subjected to a CCK-8 assay. Data are presented from three independent experiments as the mean±SD (*p<0.05). (H) ARP-1 cells were transiently transfected with Mock or ovHMGB1 vector, cultured and counted as indicated in F and growth curve was generated (*p<0.05).