Fig 2: Targeting glutaminase 1 (GLS1) inhibits M2 polarization of macrophages in the alkali-injured mouse corneas. (a) RT-PCR analysis of M1 and M2 marker genes in the alkali-injured mouse corneas treated with DMSO or CB-839 (200 mg/kg) for 7 days. (b) Immunofluorescence double staining of F4/80 (red) and M2-specific marker Mrc1 (green) in the alkali-injured mouse corneas treated with DMSO or CB-839 for 7 days. Scale bars: 50 μm. (c) Representative Western blot and quantitative analysis of STAT1, phospho-STAT1 (Tyr701), STAT3, phospho-STAT3 (Tyr705), STAT6, and phospho-STAT6 (Tyr641) in the normal corneas (control) and the alkali-injured corneas treated with DMSO or CB-839 for 7 days. β-Actin served as an endogenous reference for normalization. n = 6 − 8/group for RT-PCR or Western blotting and n = 4 − 6/group for immunofluorescence. Each experiment repeated three times, and data in graphs represent the means ± SEM. ns: no significance. ∗P < 0.05.

Fig 3: Immunohistochemistry for F4/80 (total macrophage or KCs stain) and CD163 (M2 phenotype). (A) Red fluorescence representing F4/80 and green fluorescence representing CD163. Scale bar: 2× (400 µm), 20× (200 µm). (B) Total macrophage content and CD163+ M2 macrophage quantification from the confocal images. Data were analyzed by one-way ANOVA followed by Tukey’s comparison test. Data are expressed as mean ± SEM (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Fig 4: VCP979 treatment prevents inflammatory cell infiltration and suppresses the p38-MAPK signaling pathway on day 14. (A) Representative CD4, CD8 and F4/80 staining pictures. Scale bar=50 µm. (B) The relative positive cells of the sections were determined by ImageJ software. (C) Reverse transcription-quantitative PCR analysis of mRNA levels of Arg1, CD206, iNOS, IL-1β, IL-6 and TNF-α in the heart tissues. (D) Western blotting analysis and (E) quantitative analysis of p-p38 and p38 protein levels in heart tissues from the MI/R+N.S and MI/R+VCP979. Results are shown as mean ± standard error of the mean. #P<0.05 and ##P<0.01, MI/R+N.S vs. MI/R+VCP979 group. MAPK, mitogen-activated protein kinase; iNOS, inducible nitric oxide synthase; p-, phosphorylated-; IL, interleukin; TNF, tumor necrosis factor; CD, cluster of differentiation; MI/R, myocardial ischemia/reperfusion; N.S, normal saline; ns, not significant; Arg1, arginase 1.

Fig 5: Tubular epithelial YAP activation associated with macrophage infiltration and renal MCP-1 upregulation.A Representative image of double immunofluorescent staining with YAP and F4/80 in Sham and IRI-28d kidneys. B YAP-activated tubules and F4/80-positive infiltrates were calculated from 55 randomly captured images at IRI-28d kidneys. Correlation between YAP-activated tubules and F4/80-positive infiltrates was analyzed (n = 55). Pearson correlation R value and P value are indicated. C Gene expression analysis for chemokine members including CCL and CXCL family in Mst1/2 knockout mice. Gene Expression Omnibus (GEO) datasets supply the RNA-sequencing data of GES95463. D Regulatory elements of the genes encoding MCP-1 from various mammals were reported by Kim et al.[20]. The TEAD-binding element (red bar) is located 100–400 bp upstream of the transcription start site of MCP-1. TSS transcription start site. E Representative western blot analysis of MCP-1 protein expression in kidney lysates from mice 14 and 28 days after IRI (n = 6 mice per group). Sham-operated mice was used as a control (n = 3 mice). F ELISA analysis of MCP-1 levels in serum. G, H Quantitative polymerase chain reaction analysis of MCP-1 (Ccl2) and CCR2 (Ccr2) mRNA expression in kidneys. AU arbitrary units. Scale bar = 50 µm in all images of A. All values are means ± SEM. *p < 0.05, **p < 0.01, and #p < 0.001 defined as significant. ns not significant.