Determination of CsnB effects on aggressive lymphoma cell lines. (A) Estimation of cell growth by MTS assay of SuDHL4, Karpas422, and UH3 72 hours after CsnB treatment in a range from 2.5 × 10−3 to 5 × 10−9 M. Cell growth was estimated by using the MTS assay in comparison with each cell line treated with dimethylsulfoxide (DMSO) as vehicle control. (B) NR4A1 mRNA expression analysis in SuDHL4, Karpas422, and UH3 after treatment with 10 µM CsnB or DMSO. Relative expression levels were calculated in comparison with each cell line treated with DMSO as vehicle control. Each bar represents the mean values of expression levels ± SD. (C-D) Apoptosis assays of SuDHL4, Karpas422, and UH3 24 hours after 10 µM DMSO or CsnB treatment. To determine apoptotic effects of CsnB, annexin V (C) staining and estimation of the percentage of cleaved caspase 3 by using FACS analysis with specific fluorophore-labeled peptides or antibodies and SubG1 peak (D) determination by using FACS analysis for cell cycle distribution were performed. Each bar represents the mean values of expression levels ± SD for analyses for cleaved caspase 3 and SubG1 peak. (E) NR4A1 mRNA expression analysis of NR4A1 and NR4A3 silenced SuDHL4 lymphoma cells and vector control 24 hours after 10 µM CsnB treatment. Relative expression levels were calculated in comparison of SpLKO.1 treated with DMSO as vehicle control. The 2 replicates of NR4A1-silenced SuDHL4 cells were termed SΔNR4A1-I and SΔNR4A1-II; the NR4A3-silenced SuDHL4 cells, SΔNR4A3; and SuDHL4 just containing the empty vector, SpLKO.1. Each bar represents the mean values of expression levels ± SD. (F) Annexin V staining of SuDHL4 lymphoma cells NR4A1 silenced by shRNA or SuDHL4 lymphoma cells NR4A3 silenced by shRNA and vector control 24 hours after CsnB treatment. Annexin V staining was performed by using FACS analysis with specific fluorophore-labeled peptides. The 2 replicates of NR4A1 (SΔNR4A1-I and SΔNR4A1-II) and of NR4A3 (SΔNR4A3) were silenced in SuDHL4 cells. SpLKO.1 contains the empty vector. (G) NR4A1 mRNA expression analysis of NR4A1 and NR4A3 silenced in UH3 immortalized B cells and scrambled controls (UH3 SCR) 24 hours after 10 µM CsnB treatment. Relative expression levels were calculated in comparison of UH3 SCR treated with DMSO as vehicle control. UH3 cells transfected with 2 different siRNAs targeting NR4A1 were termed UH3-siNR4A1-1 and UH3-siNR4A1-9. The siRNA targeting NR4A3 was termed UH3-siNR4A3-2. Each bar represents the mean values of expression levels ± SD. (H) Annexin V staining of UH3 cells silenced by siRNA targeting NR4A1 or NR4A3 and scrambled control 24 hours after CsnB treatment. Annexin V staining was performed by using FACS analysis with specific fluorophore-labeled peptides.
