Figure 2
Figure 2. Expression of MAPK constitutive activators differentially regulates DC maturation phenotype. BM-derived DCs were transduced with dual lentivectors and grown for 6 days at 37°C. Surface staining was performed for each DC marker. (A) Dot plots from flow cytometric analyses for CD40 in DCs transduced with the indicated lentivectors (top of each panel). CD40 APC fluorescence levels (y-axis) were represented as a function of GFP fluorescence (x-axis). Quadrants were established based on fluorescence of the isotype control in untransduced cells. Percentage of cells and mean fluorescence intensities are indicated in each quadrant. LPS represents DCs treated with LPS 24 hours before staining. (B) Dot plots from flow cytometric analyses for CD40 as shown in panel A. The 2 top panels represent DCs transduced with lentivectors encoding only GFP (left) or lentivectors encoding MEK1 ΔNES ED using the same settings as in panel A. The 2 bottom panels represent DCs transduced with the lentivectors shown in the top, but measured on a more sensitive flow cytometry (FACS) setting to visualize CD40 down-regulation on both transduced and nontransduced cells. (C) Histograms from dot plots as shown in panel A gated on GFP-positive transduced DCs. The number of events is represented as a function of log fluorescence intensity for the DC markers shown. I indicates the distribution from the isotype control. The horizontal line indicates the cells considered to be positive, which is set to exclude 95% of cells stained with the isotype control. Percentage of positive DCs and mean fluorescent intensities are indicated within each histogram, for DCs transduced with lentivectors encoding the indicated activators. The first row of histograms show untransduced DCs (UT, filled histogram), DCs transduced with a control lentivector only expressing GFP (GFP, thin line), and DCs treated with LPS (LPS, dotted line). The second row shows histograms from DCs transduced with lentivectors encoding only GFP (GFP, filled histogram), expressing MKK6EE (MKK6, thin line), or treated with LPS (dotted line). The third row shows histograms from DCs transduced with a control lentivector expressing GFP (GFP, filled histogram), expressing MKK7-JNK1 (JNK, thin line), or treated with LPS (dotted line). The fourth row shows histograms from DCs transduced with a control lentivector expressing GFP (GFP, filled histogram), expressing MEK1 ΔNES ED (MEK, thin line), or treated with LPS (dotted line).

Expression of MAPK constitutive activators differentially regulates DC maturation phenotype. BM-derived DCs were transduced with dual lentivectors and grown for 6 days at 37°C. Surface staining was performed for each DC marker. (A) Dot plots from flow cytometric analyses for CD40 in DCs transduced with the indicated lentivectors (top of each panel). CD40 APC fluorescence levels (y-axis) were represented as a function of GFP fluorescence (x-axis). Quadrants were established based on fluorescence of the isotype control in untransduced cells. Percentage of cells and mean fluorescence intensities are indicated in each quadrant. LPS represents DCs treated with LPS 24 hours before staining. (B) Dot plots from flow cytometric analyses for CD40 as shown in panel A. The 2 top panels represent DCs transduced with lentivectors encoding only GFP (left) or lentivectors encoding MEK1 ΔNES ED using the same settings as in panel A. The 2 bottom panels represent DCs transduced with the lentivectors shown in the top, but measured on a more sensitive flow cytometry (FACS) setting to visualize CD40 down-regulation on both transduced and nontransduced cells. (C) Histograms from dot plots as shown in panel A gated on GFP-positive transduced DCs. The number of events is represented as a function of log fluorescence intensity for the DC markers shown. I indicates the distribution from the isotype control. The horizontal line indicates the cells considered to be positive, which is set to exclude 95% of cells stained with the isotype control. Percentage of positive DCs and mean fluorescent intensities are indicated within each histogram, for DCs transduced with lentivectors encoding the indicated activators. The first row of histograms show untransduced DCs (UT, filled histogram), DCs transduced with a control lentivector only expressing GFP (GFP, thin line), and DCs treated with LPS (LPS, dotted line). The second row shows histograms from DCs transduced with lentivectors encoding only GFP (GFP, filled histogram), expressing MKK6EE (MKK6, thin line), or treated with LPS (dotted line). The third row shows histograms from DCs transduced with a control lentivector expressing GFP (GFP, filled histogram), expressing MKK7-JNK1 (JNK, thin line), or treated with LPS (dotted line). The fourth row shows histograms from DCs transduced with a control lentivector expressing GFP (GFP, filled histogram), expressing MEK1 ΔNES ED (MEK, thin line), or treated with LPS (dotted line).

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