Figure 5
Figure 5. Up-regulation of Jagged-2 determines the immunologic characteristics of MSC-DCs. (A) Identification of an efficient and specific siRNA targeted against Jagged-2. The MSC-DC was infected with different Jagged-2–siRNA retroviral vectors. After 5 days of transfection, total RNA was isolated and reverse transcribed, and real-time PCR was performed with Jagged-2 mRNA as the target and β-actin as an internal control. The normalized ratio of Jagged-2 to β-actin in untreated cells was set to 100% (column 1). The transfection with scrambled negative control (NC) 1 (column 2), NC2 (column 3), mock (column 4) siRNA, and Jagged-2–siRNA1 demonstrated no significant down-regulation of the Jagged-2 mRNA. A significant effect (*P < .01) on Jagged-2 mRNA was detected for Jagged-2–siRNA2 (column 6). The specific mRNA level was reduced up to 70%. The data are presented as mean (± SD) of 3 independent experiments. Changes were considered as significant if P < .01. (B) Jagged-2 knockdown is confirmed at the protein level. After 5 days of transfection, the total cellular protein of extract of DCs was separated for analysis of Jagged-2 expression. Jagged-2-siRNA–MSC-DCs showed dramatically reduced Jagged-2 protein levels compared with untreated, NC, and mock vector treated cells. The data are representative of 3 independent experiments with cells. (C) Knockdown of Jagged-2 enhances the immunogenicity while decreases the immunoregulatory function of MSC-DCs. The top panel shows that CFSE-labeled lymphocytes (H-2Kb) were cultured with maDCs (H-2Kd), MSC-DCs (H-2Kd), NC-MSC-DCs, (H-2Kd) and siRNA–MSC-DCs (H-2Kd), respectively, in the absence or presence of maDCs (H-2Kd) stimulation for 3 days, and the total number of live lymphocytes in each well was measured by flow cytometry. Data represent mean (± SD) of triplicate samples. *P < .05. In the bottom panel, FACS analysis showed that the effects of Jagged-2 RNAi on the lymphocyte proliferation. Data are representative of at least 3 independent experiments. Dotted line, unstimulated lymphocytes.

Up-regulation of Jagged-2 determines the immunologic characteristics of MSC-DCs. (A) Identification of an efficient and specific siRNA targeted against Jagged-2. The MSC-DC was infected with different Jagged-2–siRNA retroviral vectors. After 5 days of transfection, total RNA was isolated and reverse transcribed, and real-time PCR was performed with Jagged-2 mRNA as the target and β-actin as an internal control. The normalized ratio of Jagged-2 to β-actin in untreated cells was set to 100% (column 1). The transfection with scrambled negative control (NC) 1 (column 2), NC2 (column 3), mock (column 4) siRNA, and Jagged-2–siRNA1 demonstrated no significant down-regulation of the Jagged-2 mRNA. A significant effect (*P < .01) on Jagged-2 mRNA was detected for Jagged-2–siRNA2 (column 6). The specific mRNA level was reduced up to 70%. The data are presented as mean (± SD) of 3 independent experiments. Changes were considered as significant if P < .01. (B) Jagged-2 knockdown is confirmed at the protein level. After 5 days of transfection, the total cellular protein of extract of DCs was separated for analysis of Jagged-2 expression. Jagged-2-siRNA–MSC-DCs showed dramatically reduced Jagged-2 protein levels compared with untreated, NC, and mock vector treated cells. The data are representative of 3 independent experiments with cells. (C) Knockdown of Jagged-2 enhances the immunogenicity while decreases the immunoregulatory function of MSC-DCs. The top panel shows that CFSE-labeled lymphocytes (H-2Kb) were cultured with maDCs (H-2Kd), MSC-DCs (H-2Kd), NC-MSC-DCs, (H-2Kd) and siRNA–MSC-DCs (H-2Kd), respectively, in the absence or presence of maDCs (H-2Kd) stimulation for 3 days, and the total number of live lymphocytes in each well was measured by flow cytometry. Data represent mean (± SD) of triplicate samples. *P < .05. In the bottom panel, FACS analysis showed that the effects of Jagged-2 RNAi on the lymphocyte proliferation. Data are representative of at least 3 independent experiments. Dotted line, unstimulated lymphocytes.

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