Fig 2: Transcriptional effects of GSK3 knockdown in monocyte-derived GM-MØ.(A) Schematic representation of the siRNA-mediated GSK3 knockdown procedure in monocyte-derived GM-MØ. Figure 4 A was created with BioRender.com. (B) Total GSK3α, GSK3β, and MAFB protein levels in monocyte-derived GM-MØ after siRNA-mediated GSK3A and/or GSK3B silencing, as determined by western blot (upper panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of GSK3β/GAPDH and GSK3α/GAPDH protein ratios from the three independent experiments are shown (lower panel) (one-way ANOVA with Dunnett’s test: *, p<0.05). (C) Heatmap of the relative expression of the genes significantly altered after GSK3A and GSK3B knockdown in siCNT-GM-MØ (lanes A–C), siGSK3A-GM-MØ (lanes D–F), siGSK3B-GM-MØ (lanes G–I), siGSK3A/B-GM-MØ (lanes J–L). Representative genes are indicated. (D) Number of differentially expressed genes (adjp<0.05) between siGSK3A-GM-MØ, siGSK3B-GM-MØ, or siGSK3A/B-GM-MØ and siCNT-GM-MØ. Differential gene expression was assessed using DESeq2. (E) Gene set enrichment analysis (GSEA) of the genes whose expression is significantly modulated by CHIR-99021 in either GM-MØ (from Figure 1, this manuscript) or in ex vivo isolated alveolar macrophages (AMØ, see below in Figure 6) on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. (F) GSEA of the indicated gene sets (from GSE68061) on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. (G) Summary of GSEA of the indicated gene sets (from GSE188278) on the ranked comparison of siGSK3A/B-GM-MØ vs. siCNT-GM-MØ. (H) Summary of GSEA of the gene sets that define the tissue-resident monocyte and macrophage states (MoMac-VERSE) Mulder et al., 2021 on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. FDRq values are indicated only if FDRq>0.0; empty dots, not significant. (I) Summary of GSEA of the gene sets that characterize the macrophage subsets identified in severe COVID-19 (Wendisch et al., 2021; Liao et al., 2020; Grant et al., 2021) on the ranked comparison of siGSK3A/B-GM-MØ vs. siCNT-GM-MØ. FDRq values are indicated only if FDRq>0.0; empty dots, not significant. Figure 4—source data 1.PDF file containing original western blots for Figure 4B, indicating the relevant bands. Figure 4—source data 2.Original files for western blot analysis displayed in Figure 4B. Figure 4—source data 3.Protein ratios of GSK3β/GAPDH and GSK3α/GAPDH in monocyte-derived GM-MØ after siRNA-mediated GSK3A and/or GSK3B silencing from the three independent experiments and statistical analysis.