Figure 2
Figure 2. Functional characterization of NR4A1 in SuDHL4 lymphoma cells. Transduced SuDHL4 cells carrying empty vector (SpLVX) or the inducible NR4A1 construct (SN1 I-III) were cultured in the presence of doxycycline (DOX), no NR4A1 induction, or absence of doxycycline (No DOX), induction of NR4A1. (A) NR4A1 mRNA expression analysis after induction. Relative expression levels were calculated in comparison with Splvx cultured with doxycycline-containing media. Each bar represents the mean values of expression levels ± SD. * indicates a significant induction of NR4A1 compared with vector control and uninduced counterpart (P < .01). (B) Western blot analysis of NR4A1 expression with and without its induction in SuDHL4 carrying the inducible NR4A1 construct. GAPDH served as loading control. (C-F, H) Apoptotic and cell growth assay of transduced SuDHL4 carrying the empty vector (Splvx) or the inducible NR4A1 construct SuDHL4 (SN1 I-III) with and without doxycycline. To determine the apoptotic effects of NR4A1 in aggressive lymphoma cells, caspase 3/7 activity (C), caspase 3 cleavage (E), SubG1 peak (F), and annexin V (H) were estimated. Furthermore, cell growth analysis was performed by employing MTS assays (D). The caspase 3/7 activity and the MTS were calculated in comparison with Splvx lymphoma cells cultured with doxycycline-containing media. Annexin V staining and cleaved caspase 3 percentage were estimated by using fluorescence-activated cell sorter (FACS) analysis with specific fluorophore-labeled peptides or antibodies. SubG1 peaks were determined by cell cycle analysis using FACS analysis. * indicates significantly higher apoptotic activity or reduced cell growth compared with vector control and uninduced counterpart (P < .01). (G) mRNA expression levels of potential NR4A target genes of transduced SuDHL4 carrying the empty vector (Splvx) or the inducible NR4A1 construct (SN1 I-III) with and without doxycycline. * indicates significantly higher induction of TRAIL, BIM 1,6, and Puma to vector control and uninduced counterpart (P < .01). (I) Visual inspection of SuDHL4 lymphoma formation with an inducible NR4A1 construct (left flank) and vector controls (right flank) with and without doxycycline administration in the NSG xenograft model after 20 days. (J) Macroscopic analysis of tumors from SuDHL4 lymphoma cells carrying the inducible NR4A1 construct (left flank) and empty vector (right flank) with and without doxycycline administration after 20 days. (K) Estimation of tumor volume by using an ultrasonic device after 20 days. * indicates significant induction of NR4A1 compared with vector control and uninduced counterpart (P < .01). (L) Histologic analysis of tumors from SuDHL4 lymphoma cells carrying empty vector (upper row) or the inducible NR4A1 construct (lower row) and with (left column) and without doxycycline (right column) administration after 20 days (magnification ×100).

Functional characterization of NR4A1 in SuDHL4 lymphoma cells. Transduced SuDHL4 cells carrying empty vector (SpLVX) or the inducible NR4A1 construct (SN1 I-III) were cultured in the presence of doxycycline (DOX), no NR4A1 induction, or absence of doxycycline (No DOX), induction of NR4A1. (A) NR4A1 mRNA expression analysis after induction. Relative expression levels were calculated in comparison with Splvx cultured with doxycycline-containing media. Each bar represents the mean values of expression levels ± SD. * indicates a significant induction of NR4A1 compared with vector control and uninduced counterpart (P < .01). (B) Western blot analysis of NR4A1 expression with and without its induction in SuDHL4 carrying the inducible NR4A1 construct. GAPDH served as loading control. (C-F, H) Apoptotic and cell growth assay of transduced SuDHL4 carrying the empty vector (Splvx) or the inducible NR4A1 construct SuDHL4 (SN1 I-III) with and without doxycycline. To determine the apoptotic effects of NR4A1 in aggressive lymphoma cells, caspase 3/7 activity (C), caspase 3 cleavage (E), SubG1 peak (F), and annexin V (H) were estimated. Furthermore, cell growth analysis was performed by employing MTS assays (D). The caspase 3/7 activity and the MTS were calculated in comparison with Splvx lymphoma cells cultured with doxycycline-containing media. Annexin V staining and cleaved caspase 3 percentage were estimated by using fluorescence-activated cell sorter (FACS) analysis with specific fluorophore-labeled peptides or antibodies. SubG1 peaks were determined by cell cycle analysis using FACS analysis. * indicates significantly higher apoptotic activity or reduced cell growth compared with vector control and uninduced counterpart (P < .01). (G) mRNA expression levels of potential NR4A target genes of transduced SuDHL4 carrying the empty vector (Splvx) or the inducible NR4A1 construct (SN1 I-III) with and without doxycycline. * indicates significantly higher induction of TRAIL, BIM 1,6, and Puma to vector control and uninduced counterpart (P < .01). (I) Visual inspection of SuDHL4 lymphoma formation with an inducible NR4A1 construct (left flank) and vector controls (right flank) with and without doxycycline administration in the NSG xenograft model after 20 days. (J) Macroscopic analysis of tumors from SuDHL4 lymphoma cells carrying the inducible NR4A1 construct (left flank) and empty vector (right flank) with and without doxycycline administration after 20 days. (K) Estimation of tumor volume by using an ultrasonic device after 20 days. * indicates significant induction of NR4A1 compared with vector control and uninduced counterpart (P < .01). (L) Histologic analysis of tumors from SuDHL4 lymphoma cells carrying empty vector (upper row) or the inducible NR4A1 construct (lower row) and with (left column) and without doxycycline (right column) administration after 20 days (magnification ×100).

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