Additional file 1 of STAT3 blockade ameliorates LPS-induced kidney injury through macrophage-driven inflammation
Creators: name: Lee, Song-Hee
name: Kim, Kyu Hong
name: Lee, Seong Min
name: Park, Seong Joon
name: Lee, Sunhwa
name: Cha, Ran-Hui
name: Lee, Jae Wook
name: Kim, Dong Ki
name: Kim, Yon Su
name: Ye, Sang-Kyu
name: Yang, Seung Hee
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Environmental Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Marine Biology

description: Supplementary Material 1: Figure S1. Representative IHC images of pSTAT3 in kidney tubules and transcriptomic analysis of time-dependent L-AKI mice. (A) pSTAT3 staining of the L-AKI model following a time-dependent manner (0, 6, 12, and 24 h) is shown. (B) The expression of pSTAT3 decreased in the LPS + Stattic group compared to that in the LPS group. Scale bar, 75 μm (200X). (C) The mRNA expression of kidney injury marker, pro-inflammatory, STAT3-associated genes in time-dependent L-AKI mice. Figure S2. Top 10 KEGG pathways and GO enrichment analyses for mice treated with LPS and Stattic. (A, B) Top 10 global KEGG pathways and GO enrichment analyses were conducted for both downregulated (A) and upregulated (B) genes. The results are visualized in dot plots, where the dot size corresponds to the gene count, and the color gradient represents the p-value . Figure S3. Common genes derived from JAK-STAT-dependent inflammatory response pathways. (A) Common genes across three distinct inflammatory response pathways were identified using KEGG pathway analysis. (B) A heatmap was constructed to visualize macrophage-related genes, specifically highlighting differences in macrophage cytokine production, activation, and migration. Figure S4. KEGG pathway analysis on the three distinct inflammatory response pathways. KEGG pathway analysis was performed on three distinct inflammatory response pathways: JAK-STAT, TLR, and TNF, represented in blue, red, and green, respectively. Additionally, 3 genes common to all pathways were further analyzed and visualized using the KEGG color map. Figure S5. KEGG pathway analysis of the response to LPS and macrophage-related genes. (A) Color maps depicting the 11 intersecting genes identified between the response to LPS and macrophage-related gene pathways, focusing on the cytokine–cytokine receptor interaction and chemokine signaling pathways. (B) Detailed KEGG pathway analysis of thermogenesis highlighting upregulated gene interactions. Figure S6. JAK-STAT signaling pathway-related upregulated genes and KEGG pathway analysis. (A) A heatmap represented JAK-STAT signaling pathway-related upregulated genes (n  = 4). (B) A dot plot for KEGG analysis of upregulated and downregulated genes was shown. (C) A table lists IL-6-mediated STAT3 signaling pathway-related genes identified from the KEGG pathway analysis. (D) A diagram from the KEGG mapper indicated both canonical (gray line) and noncanonical (red line) STAT signaling pathways, along with upregulated (red) and downregulated (green) genes. (E) A network analysis of the IL-6-mediated STAT3 signaling pathway was derived from (C). Figure S7. Network analysis of GO terms related to LPS and macrophage genes. A comprehensive network analysis of LPS and macrophage-related genes was constructed using Cytoscape software. Figure S8. Systemic changes in the spleens of L-AKI mice using real-time qPCR. The expression levels of (A) proinflammatory genes and (B) macrophage-related and STAT3-associated genes were analyzed in the spleens of L-AKI mice (n  = 8 in each group). The results are presented as mean ± SEM, and statistical analysis was conducted using an unpaired two-tailed Student’s t-test. * P  &lt; 0.05, ** P  &lt; 0.01, *** P  &lt; 0.001. Figure S9. pSTAT3-positive cells of LPS-stimulated RAW264.7 cells. Histogram and quantification graph of pSTAT3-positive cells using FACS analysis after (A) time-dependent, (B) dose-dependent LPS treatment, and (C) Stattic treatment with LPS stimulation in RAW264.7 cells.