S1PR2 expression is regulated by the TGF-β/SMAD signaling pathway. (A) S1PR2 expression after 24 hours of treatment with the indicated increasing doses of TGF-β, as assessed in the SU-DHL-6, Oci-Ly10, RC-K8, and Oci-Ly3 DLBCL cell lines by qRT-PCR. (B) The DLBCL cell line SU-DHL-6 was treated with FOXP1 targeting siRNA for 48 hours and subjected to treatment with 2 ng/mL TGF-β for an additional 24 hours. (A-B) Data are pooled from 3 or more independent experiments. Graphs show mean ± SEM; P values were calculated using the Student t test. (C) The indicated DLBCL cell lines were treated with 2 ng/mL TGF-β for 1 hour and subjected to immunoblotting with antibodies against the indicated SMAD proteins, p-SMAD1/5/9 and tubulin. Representative immunoblots of at least 2 independent experiments are shown. (D) TGF-βR2 surface expression of the indicated DLBCL cell lines, as assessed by flow cytometry. The plots are representative for 2 independent experiments. (E) pSMAD1/5/9 ChIP of cells treated or not with 5 ng/mL TGF-β for 4 hours; an unspecific rbIgG antibody was used as control. Eluted DNA was subjected to PCR using primers amplifying 2 regions 2.5 and 5 kb upstream of the S1PR2 TSS. MyoD was amplified as negative control; CDKN1A and ID1 were used as positive controls for canonical TGF-β and BMP signaling. Graphs represent the fold change of the yield relative to 1% input of the pSMAD1/5/9 sample vs rbIgG; means ± SD of 2 independent experiments are shown. *P < .05; **P < .01; ***P < .001; ****P < .0001.