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Figure 1 Variation in the temporal expression of IRF7 during treatment with imiquimod as determined by real time RT-PCR. 'AK,' designates pretreatment AK lesions. 'WK1 IMIQ', 'WK2 IMIQ' and 'WK4 IMIQ' designate treatment times week 1, 2 and 4. '4 WK Post designates' 4 weeks post end of treatment (WK4 treatment). Fold change was calculated with respect to sun-unexposed, non-lesional skin. S-01, S-02, S-06, S-15 and S-16 are samples from subjects 1, 2, 6, 15 and 16.
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Figure 2 Comparison of gene expression data obtained by Affymetrix GeneChip analysis and real time RT-PCR. Linear regression analysis for IRF7 comparing fold change values for individual subjects as measured by Affymetrix analysis and real time RT-PCR analysis. The imiquimod response fold change, which is the maximum response from week 1, week 2 and week 4 treatment times was used. Fold change was calculated relative to sun-unexposed, nonlesional skin samples for 13 subjects treated with imiquimod. The R square value for the comparison was 0.83.
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Figure 3 Basal TLR, IRF7, and MyD88 gene expression in skin biopsies as determined by real time RT-PCR. White bars represent pretreatment AK, black bars represent during imiquimod treatment (maximum response value from week 1, week 2 and week 4 treatment times), and hatched bars represent 4-weeks post treatment. Relative copy number was determined as outlined in Methods and Materials section. Asterisks indicate those genes that had p-values 2].
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Figure 4 Increase in expression of the helicase family of virus-sensing genes upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)DDX58 (RIG-I) and (B)IFIH1 (MDA5). Experimental conditions are as described in the Methods and Materials section. Box plots were generated using MINITAB version 14. 'AK,' 'IMIQ,' and 'Post' designate pretreatment AK, imiquimod-treated skin during treatment (maximum response from week 1, week 2 and week 4), and imiquimod-treated skin 4 weeks post treatment, respectively. Fold change was calculated with respect to sun-unexposed skin. Boxes indicate the median 95% confidence intervals, asterisks designate outliers, and the lines connect median values. P values are given for 2-way ANOVA, comparing the fold change values for pretreatment AK samples and imiquimod-treated samples (n = 13).
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Figure 5 Cluster analysis of genes regulated by imiquimod treatment. Two-way hierarchical clustering was performed as described in the Methods and Materials section. 'AK,' 'IMIQ,' and 'Placebo' designate the fold change with respect to sun-unexposed, nonlesional skin for pretreatment AK samples; for samples during imiquimod treatment (maximum response from week 1, week 2 and week 4), and samples for vehicle-treatment (maximum response from week 1, week 2 and week 4) respectively. Numbers designate subjects. Hierarchical clustering was performed using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. Red, white, and green indicate up-regulated, unchanged, and down-regulated genes, respectively. The color bar insert shows the corresponding expression levels. The cluster consists of 530 imiquimod-responsive genes whose expression was statistically different when comparing the AK group of samples to the IMIQ group of samples. Expression changes for the 530 genes are documented in [Additional file 1].
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Figure 6 Cluster of 46 immune response genes that are also known to be inducible upon treatment with type 1 interferons as reported in [18, 43, 45, 48, 51, 102] Cluster analysis was performed as described in Figure 5. [See Additional file 3].
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Figure 7 Imiquimod treatment is associated with an increase in expression of pro-apoptotic genes as determined by Affymetrix GeneChip analysis. (A) TNFSF10 (B)MX1. Experimental conditions and analysis are as described for Figure 4.
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Figure 8 Increase in expression of chemokines after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CXCL10 (IP10), (B)CXCL11 (ITAC). Experimental conditions and analysis are as described for Figure 4.
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Figure 9 Increase in expression of genes indicating the infiltration of DCs upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CD86 (B) ILT7. Experimental conditions and analysis are as described for Figure 4.
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Figure 10 Increase in expression of genes associated with NK cells, and cytotoxic T cells after treatment with imiquimod as determined by Affymetrix gene expression. (A)GZMA (B) NKG7. Experimental conditions and analysis are as described for Figure 4.
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Figure 11 Increase in expression of genes associated with lymphocyte function after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A) SELL (CD62L) (B) NT5E (DC73). Experimental conditions and analysis are as described for Figure 4.
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