TGF-β induces S1PR2-dependent apoptosis in DLBCL cell lines in vitro and in vivo. (A) Cell viability and apoptosis, as determined by Cell Titer Blue assay and Annexin V staining, of the indicated cell lines after 24 hours of exposure to increasing concentrations of TGF-β; values are normalized to the untreated control sample. (B) The DLBCL cell line SU-DHL-6 was treated with FOXP1 targeting siRNA for 48 hours, subjected to 2 ng/mL TGF-β for additional 24 hours, and analyzed as shown in panel A. (A-B) Data are pooled from 3 or more independent experiments. Graphs show means ± SEM; P values were calculated using the Student t test. (C) Three S1PR2^+/+, 1 S1PR2^+/−, and 3 S1PR2^−/− clones generated in the SU-DHL-6 cell line were treated with 2 ng/mL TGF-β and analyzed for apoptosis by Annexin V staining. Bars represent pooled data for each genotype relative to the untreated control of each clone. Each clone was analyzed 3 to 6 times. Graphs represent means ± SEM; P values were calculated using the Student t test. (D-F) Ten million SU-DHL-6 cells were injected subcutaneously into both flanks of NSG mice. (D) One tumor per mouse was injected intratumorally with TGF-β at the depicted intervals; the other received vehicle only. Tumor volumes were measured (E) after excision and (F) RNA was extracted and qRT-PCR for S1PR2 was performed on excised tumor tissue. (F) Each dot represents 1 tumor and results are pooled from 2 independent experiments. S1PR2 expression analysis was performed in only 1 of the 2 studies with n = 10 per group. Two control and 1 TGF-β–treated tumor had to be excluded because of insufficient RNA quality. TGF-β–treated and control tumors are compared for each mouse. P values were calculated using the Mann-Whitney U test. *P < .05; **P < .01; ***P < .001; ****P < .0001.