Figure 6.
Figure 6. TGF-β signaling via TGF-βR2 and SMAD1 activates S1PR2 expression and induces apoptosis of DLBCL cells and SMAD1 expression is downregulated in DLBCL patients. (A) Three SMAD1+/+ and 2 SMAD1−/− as well as 3 TGFβR2+/+ and 3 TGFβR2−/− clones (all generated in the SU-DHL-6 cell line) were treated with 2 ng/mL TGF-β for 24 hours and analyzed for apoptosis with Annexin V staining. Bars represent pooled data for each genotype relative to the untreated control of each clone. (B) The same clones as in panel A were subjected to RNA extraction and S1PR2-specific qRT-PCR. (A-B) Each clone was analyzed twice; graphs represent means ± SEM; P values were calculated using the Student t test. (C) The DLBCL cell line SU-DHL-6 was treated with SMAD1-targeting siRNA for 48 hours and subjected to 2 ng/mL TGF-β for additional 24 hours. Cells were analyzed for apoptosis by Annexin V staining. Graphs show pooled results from 6 independent experiments. Means ± SEM are represented. P values were calculated using the Student t test. (D) Absolute cell counts of 2 to 3 independent clones derived from FACS single cells of the indicated genotypes were compared under standard cell culture conditions over 10 days without medium change. Two experimental replicates are shown. P values were calculated using the Student t test on the average value for each genotype. (E) Ten million cells each of 3 SMAD1+/+ clones (gray) and 2 SMAD1−/− clones (orange, in SU-DHL-6) were injected subcutaneously into the flanks of NSG mice. Tumors were excised and tumor weights and tumor volumes were determined at the study end point 24 days after injection. Every dot represents 1 tumor; plots show pooled data from 2 independent experiments. (F) Ten million cells of 6 SMAD1/TGFβR2+/+ clones (gray) and 2 SMAD1−/− (orange) or 3 TGFβR2−/− (blue, all in SU-DHL-6) clones were injected IV into MISTRG mice. Mice were euthanized 35 days postinjection, their spleens were weighed, and the frequencies of hCD45+ cells in the spleens and bone marrow was determined by flow cytometry. Every dot represents 1 mouse; graphs represent data from 3 experiments. (E-F) Horizontal lines indicate medians; P values were calculated using the Mann-Whitney U test. (G) Negative (left) and positive (right) SMAD1 immunohistochemical staining of DLBCL patient samples. Size bar represents 20 μm. *P < .05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

TGF-β signaling via TGF-βR2 and SMAD1 activates S1PR2 expression and induces apoptosis of DLBCL cells and SMAD1 expression is downregulated in DLBCL patients. (A) Three SMAD1+/+ and 2 SMAD1−/− as well as 3 TGFβR2+/+ and 3 TGFβR2−/− clones (all generated in the SU-DHL-6 cell line) were treated with 2 ng/mL TGF-β for 24 hours and analyzed for apoptosis with Annexin V staining. Bars represent pooled data for each genotype relative to the untreated control of each clone. (B) The same clones as in panel A were subjected to RNA extraction and S1PR2-specific qRT-PCR. (A-B) Each clone was analyzed twice; graphs represent means ± SEM; P values were calculated using the Student t test. (C) The DLBCL cell line SU-DHL-6 was treated with SMAD1-targeting siRNA for 48 hours and subjected to 2 ng/mL TGF-β for additional 24 hours. Cells were analyzed for apoptosis by Annexin V staining. Graphs show pooled results from 6 independent experiments. Means ± SEM are represented. P values were calculated using the Student t test. (D) Absolute cell counts of 2 to 3 independent clones derived from FACS single cells of the indicated genotypes were compared under standard cell culture conditions over 10 days without medium change. Two experimental replicates are shown. P values were calculated using the Student t test on the average value for each genotype. (E) Ten million cells each of 3 SMAD1+/+ clones (gray) and 2 SMAD1−/− clones (orange, in SU-DHL-6) were injected subcutaneously into the flanks of NSG mice. Tumors were excised and tumor weights and tumor volumes were determined at the study end point 24 days after injection. Every dot represents 1 tumor; plots show pooled data from 2 independent experiments. (F) Ten million cells of 6 SMAD1/TGFβR2+/+ clones (gray) and 2 SMAD1−/− (orange) or 3 TGFβR2−/− (blue, all in SU-DHL-6) clones were injected IV into MISTRG mice. Mice were euthanized 35 days postinjection, their spleens were weighed, and the frequencies of hCD45+ cells in the spleens and bone marrow was determined by flow cytometry. Every dot represents 1 mouse; graphs represent data from 3 experiments. (E-F) Horizontal lines indicate medians; P values were calculated using the Mann-Whitney U test. (G) Negative (left) and positive (right) SMAD1 immunohistochemical staining of DLBCL patient samples. Size bar represents 20 μm. *P < .05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

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