Figure 1
Figure 1. Expression and functional activity of the IL-21/IL-21R system in HRS cell lines. (A) RT-PCR analyses of IL21 and IL21R mRNA expression in Hodgkin (L428, L1236, KM-H2, L591, HDLM-2, L540, L540Cy) and non-Hodgkin (Reh, Namalwa, SU-DHL-4) cell lines and purified CD19+ tonsillar B cells. Expression of GAPDH was analyzed as a control. (B) Protein expression of IL-21 and IL-21R in various cell lines. Top panels show protein expression of IL-21 in the indicated cell lines, which was analyzed by Western blot in whole-cell extracts. Expression of β-actin was analyzed as a control. Bottom panels show analysis of IL-21R expression by flow cytometry in HRS (L428, L1236, KM-H2, L591, HDLM-2, L540) and non-Hodgkin (Reh, Namalwa, SU-DHL-4) cell lines. IC indicates isotype control. (C) Induction of STAT3 activity in L428 cells following treatment with rhIL-21. L428 cells were cultured for 12 hours in medium containing 0.5% FCS. Thereafter, cells were stimulated with 100 ng/mL rhIL-21, and whole-cell extracts were prepared at the indicated times. Extracts were analyzed by Western blot for phospho-STAT3 (pSTAT3) and, as a control, STAT3 expression (top panels). STAT3 DNA-binding activity was analyzed in whole-cell extracts by EMSA (bottom panel). (D) Inhibition of IL-21 by IL-21R:Fc. Top panels: to verify functionality of IL-21R:Fc, HRS L428 cells were left untreated or treated for 15 minutes with 20 ng/mL rhIL-21 preincubated with BSA (carrier for the :Fc constructs), or with 30 μg/mL of the control IgG1:Fc or the specific IL-21R:Fc construct. Thereafter, whole-cell extracts were analyzed by Western blot for pSTAT3 and STAT3 expression. Bottom panels: KM-H2 cells with constitutive STAT3 phosphorylation were left untreated, incubated with BSA, or treated with 50 μg/mL IgG1:Fc (control) or IL-21R:Fc, as indicated. After 6 hours, nuclear extracts were prepared and analyzed by Western blot for pSTAT3 and, as a control, PARP-1 expression. (E) Regulation of STAT3 target genes following stimulation of HRS cell lines with rhIL-21. Top panels: L428 HRS cells were stimulated with rhIL-21 as described in panel D. At the indicated times, mRNA was prepared and analyzed by RT-PCR for expression of IL6, MCL1, and, as a control, GAPDH. Bottom panel: measurement of secreted IL-6 by ELISA following stimulation with rhIL-21. The cell lines L428, L1236, HDLM-2, and KM-H2 were cultured in 1% FCS and stimulated every 8 hours with rhIL-21. After 24 hours, supernatants were analyzed by an IL-6–specific ELISA. The amount of IL-6 in the supernatants is shown in nanograms per milliliter. As controls, standard medium and the reagent diluent for the standard (reagent D) were included. Error bars denote SD. (F) KM-H2 cells were treated as described in panel D. After 6 hours, mRNA was prepared and analyzed by RT-PCR for expression of IL6, MCL1, and, as a control, GAPDH.

Expression and functional activity of the IL-21/IL-21R system in HRS cell lines. (A) RT-PCR analyses of IL21 and IL21R mRNA expression in Hodgkin (L428, L1236, KM-H2, L591, HDLM-2, L540, L540Cy) and non-Hodgkin (Reh, Namalwa, SU-DHL-4) cell lines and purified CD19+ tonsillar B cells. Expression of GAPDH was analyzed as a control. (B) Protein expression of IL-21 and IL-21R in various cell lines. Top panels show protein expression of IL-21 in the indicated cell lines, which was analyzed by Western blot in whole-cell extracts. Expression of β-actin was analyzed as a control. Bottom panels show analysis of IL-21R expression by flow cytometry in HRS (L428, L1236, KM-H2, L591, HDLM-2, L540) and non-Hodgkin (Reh, Namalwa, SU-DHL-4) cell lines. IC indicates isotype control. (C) Induction of STAT3 activity in L428 cells following treatment with rhIL-21. L428 cells were cultured for 12 hours in medium containing 0.5% FCS. Thereafter, cells were stimulated with 100 ng/mL rhIL-21, and whole-cell extracts were prepared at the indicated times. Extracts were analyzed by Western blot for phospho-STAT3 (pSTAT3) and, as a control, STAT3 expression (top panels). STAT3 DNA-binding activity was analyzed in whole-cell extracts by EMSA (bottom panel). (D) Inhibition of IL-21 by IL-21R:Fc. Top panels: to verify functionality of IL-21R:Fc, HRS L428 cells were left untreated or treated for 15 minutes with 20 ng/mL rhIL-21 preincubated with BSA (carrier for the :Fc constructs), or with 30 μg/mL of the control IgG1:Fc or the specific IL-21R:Fc construct. Thereafter, whole-cell extracts were analyzed by Western blot for pSTAT3 and STAT3 expression. Bottom panels: KM-H2 cells with constitutive STAT3 phosphorylation were left untreated, incubated with BSA, or treated with 50 μg/mL IgG1:Fc (control) or IL-21R:Fc, as indicated. After 6 hours, nuclear extracts were prepared and analyzed by Western blot for pSTAT3 and, as a control, PARP-1 expression. (E) Regulation of STAT3 target genes following stimulation of HRS cell lines with rhIL-21. Top panels: L428 HRS cells were stimulated with rhIL-21 as described in panel D. At the indicated times, mRNA was prepared and analyzed by RT-PCR for expression of IL6, MCL1, and, as a control, GAPDH. Bottom panel: measurement of secreted IL-6 by ELISA following stimulation with rhIL-21. The cell lines L428, L1236, HDLM-2, and KM-H2 were cultured in 1% FCS and stimulated every 8 hours with rhIL-21. After 24 hours, supernatants were analyzed by an IL-6–specific ELISA. The amount of IL-6 in the supernatants is shown in nanograms per milliliter. As controls, standard medium and the reagent diluent for the standard (reagent D) were included. Error bars denote SD. (F) KM-H2 cells were treated as described in panel D. After 6 hours, mRNA was prepared and analyzed by RT-PCR for expression of IL6, MCL1, and, as a control, GAPDH.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal