*3.4. External Validation in Public Studies of mRNA Expression*

Because we could not find a benchmark study on blood protein-based drug responsiveness to antidepressants, we examined the expression patterns of LMM-significant 37 proteins described in the results of large-scale studies at the blood circulating cell-free mRNA level from two publicly available GEO datasets—(GSE146446 [26] and GSE45468 [27]). Unlike the proteomic study above, the two GEO studies contained results on the effects of patients receiving a placebo. In the first GSE146446 dataset, mRNA expression in the blood of 171 depressed patients was studied, and patients' responses to an antidepressant vs. the placebo were monitored. The antidepressant used was duloxetine. These data contain quantitative mRNA expressions in patients before and after 8 weeks of taking the antidepressant and placebo. There were 96 patients who received the drug, including 75 responders and 21 non-responders; and 107 patients received the placebo, including 44 responders and 63 non-responders. The 37 plasma proteins that were significant in time and response were all found in the dataset, and these were analyzed by LMM. Among them, *MYH9* represented significance for the treatment/response/time interaction term, *PCSK9* showed significance for the treatment/response interaction term (*p*-value < 0.05), and *PLEK* showed significance for treatment/response and treatment/time/response interaction terms (*p*-value < 0.05; Figure 3A). The second GSE45468 dataset reflected blood mRNA expression in 52 patients. These data included mRNA expression in patients before and after 6 h, 24 h, and 2 weeks of infusion of infliximab and a placebo. There were 23 patients who received the drug, including 12 responders and 11 non-responders, and there were 15 responders and 14 non-responders among 29 patients who received the placebo. In this dataset, only 13 out of 37 proteins were found and subjected to LMM analysis. Among them, *CALU* represented significance for the treatment/time/response interaction term (*p*-value < 0.05), and *CTSD* and *SH3BGRL3* represented significance for the treatment/response interaction term (*p*-value < 0.05; Figure 3B).

**Figure 3.** External validation of LMM-significant 37 proteins in two public GEO datasets (GSE146446 and GSE45468). (**A**) In the GSE146446 dataset, the quantitative mRNA expression changes in three genes, *MYH9*, *PCSK9*, and *PLEK*, before and after 8 weeks of taking the antidepressant and placebo in responders (blue color) and non-responders (red color). Error bars represent standard error of the mean. (**B**) In the GSE45468 dataset, the mRNA expression level of patients for three genes, *CALU*, *CTSD* and *SH3BGRL3*, before infliximab infusion and after 6 h, 24 h, and 2 weeks is shown in box plots. Responders are shown in blue color and non-responders are shown in red color.
