Fig 2: WEE1 activation mediates oxLDL‐induced inflammatory response in macrophages. A) MPMs isolated from WEE1f/f and WEE1MCKO mice were challenged with oxLDL (50 µg mL−1) for 6 h. Total RNA was sequenced to identify differentially expressed genes (DEGs). Volcano plot analysis of DEGs up‐regulated (red) and down‐regulated (blue) in WT+oxLDL group compared to WT group or in KO+oxLDL group compared to WT+oxLDL group. FC, fold change. B) GO enrichments of DEGs in WT+oxLDL group compared to WT group or in KO+oxLDL group compared to WT+oxLDL group. C) Heat map of TOP30 DEGs associated with inflammatory response in KO+oxLDL group compared to WT+oxLDL group. D–F) MPMs isolated from WEE1f/f and WEE1MCKO mice were challenged with oxLDL (50 µg mL−1) for 6 h. mRNA levels of inflammatory factors D) and proinflammatory chemokines E,F) were determined via RT‐qPCR (n = 3). The values were normalized to Actb. G,H) MPMs isolated from WEE1f/f and WEE1MCKO mice were challenged with oxLDL (50 µg mL−1) for 24 h. Protein levels of TNF‐α G) and IL‐6 H) were analyzed using ELISA (n = 3). I,J) MPMs transfected with WEE1 or empty vector (EV) were challenged with oxLDL (50 µg mL−1) for 24 h. Protein levels of TNF‐α I) and IL‐6 J) were analyzed using ELISA (n = 3). K,L) MPMs isolated from WEE1MCKO mice were transfected with WEE1S642E or WEE1S642A for 24 h. Protein levels of TNF‐α K) and IL‐6 L) were analyzed using ELISA (n = 3). M) The chemical structure of MK1775. N.O) MPMs were pretreated with MK1775 (5 and 10 µm) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 µg mL−1) for 24 h. Protein levels of TNF‐α N) and IL‐6 O) were analyzed using ELISA (n = 3). P) MPMs were pretreated with MK1775 (5 and 10 µm) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 µg mL−1) for 6 h. mRNA levels of Tnf‐α and Il‐6 were determined via RT‐qPCR (n = 3). The values were normalized to Actb. Data were shown as mean ± SEM. D–F and P, two‐way ANOVA followed by Tukey's test; G‐J and N,O, one‐way ANOVA followed by Tukey's test; K,L, Student's t‐test.

Fig 3: Macrophage p‐WEE1 is up‐regulated in atherosclerotic plaques. A) Venn diagram representing kinase enrichment analysis in atherosclerotic aortas compared to normal aortas from 4 public mouse datasets (GSE21419, GSE213303, GSE163657, and GSE205929). B,C) Western blot analysis B) and densitometric quantification C) of p‐WEE1, p‐SGK1, p‐AURKB, p‐ARAF, p‐PRKD3 in aortas of LFD and HFD‐fed ApoE−/− mice. GAPDH, WEE1, SGK1, AURKB, ARAF and PRKD3 were used as loading controls (n = 6). D,E) Western blot analysis D) and densitometric quantification E) of p‐WEE1 in human non‐atherosclerotic carotid artery tissues (NAS) or atherosclerotic carotid artery tissues (AS) from three atherosclerotic patients. GAPDH and WEE1 were used as loading controls (n = 3). F,G) Representative immunohistochemistry staining F) and quantification G) of p‐WEE1 in NAS or AS from three atherosclerotic patients (n = 3, scale bar = 25 µm). H,I) Representative immunofluorescence staining H) and quantification I) of F4/80 (green) or α‐SMA (green) and p‐WEE1 (red) in aortic roots (n = 6, scale bar = 50 µm). J) Western blot analysis and densitometric quantification of WEE1 in mouse aortic vascular smooth muscle cell (MOVAS), human umbilical vein endothelial cell (HUVEC), and mouse primary peritoneal macrophage (MPM). GAPDH was used as the loading control (n = 3). K) Time‐course of p‐WEE1 induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs were exposed to oxLDL (50 µg mL−1) for indicated time. Western blot analysis and densitometric quantification of p‐WEE1 were shown. GAPDH was used as the loading control (n = 3). Data were shown as mean ± SEM. C, E, G, and I, Student's t‐test; J,K, one‐way ANOVA followed by Tukey's test.

Fig 4: WEE1 mediates inflammatory response via phosphorylating p65 at S536 in macrophages. A) NF‐κB signaling pathway is enriched in gene‐set enrichment analysis (GSEA) of KO+oxLDL versus WT+oxLDL group. B,C) MPMs isolated from WEE1f/f and WEE1MCKO mice were challenged with oxLDL (50 µg mL−1) for 1 h. Western blot analysis B) and densitometric quantification C) of p‐p65, IκBα and p‐IKKβ. GAPDH, p65, and IKKβ were used as loading controls (n = 3). D) MPMs were treated as described in panel B). Western blot analysis and densitometric quantification of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n = 3). E,F) MPMs were treated as described in panel B). Representative immunofluorescence staining images E) and quantification F) of p65 (red) translocating into nucleus in MPMs. Cells were counterstained with DAPI (blue). Scale bar = 25 µm, n = 3. PCC, Pearson correlation coefficient. G,H) Western blot analysis G) and densitometric quantification H) of p‐p65 in aortic tissues of the mice. GAPDH and p65 were used as loading controls (n = 6). I,J) Representative immunofluorescence staining images I) and quantification J) of p‐p65 (red) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar = 50 µm, n = 6. Plaques were analyzed from first appearance of intact three sinus valves. K,L) MPMs transfected with WEE1 or EV were challenged with oxLDL (50 µg mL−1) for 1 h. Western blot analysis K) and densitometric quantification L) of p‐p65. GAPDH and p65 were used as loading controls (n = 3). M,N) MPMs isolated from WEE1MCKO mice were transfected with WEE1S642E or WEE1S642A for 24 h. Western blot analysis M) and densitometric quantification N) of p‐p65. GAPDH and p65 were used as loading controls (n = 3). O,P) MPMs isolated from WEE1MCKO mice were transfection with WEE1S642E and then treated with PTD‐p65 peptide (100 µm) or vehicle (DMSO, 1‰) for 24 h. Protein levels of TNF‐α O) and IL‐6 P) were analyzed using ELISA (n = 3). Q) MPMs were treated as described in panel O,P). Western blot analysis of p‐p65. GAPDH and p65 were used as loading controls (n = 3). R) MPMs were pretreated with MK1775 (5 and 10 µm) or vehicle (DMSO, 1‰) for 1 h, followed by exposure of oxLDL (50 µg mL−1) for 1 h. Western blot analysis of p‐p65, IκBα, and p‐IKKβ. GAPDH, p65, and IKKβ were used as loading controls (n = 3). S) MPMs were treated as described in panel R). Western blot analysis and densitometric quantification of p65 in nucleus and cytoplasm. GAPDH was used as the loading control for cytosolic fractions. Lamin B1 was used as the loading control for nuclear fractions (n = 3). T) Working model of WEE1 modulating NF‐κB signaling pathway. Data were shown as mean ± SEM. C,D, two‐way ANOVA followed by Tukey's test; F, L, and O,P, one‐way ANOVA followed by Tukey's test; H, J, and N, Student's t‐test.

Fig 5: Pharmacological inhibitor of WEE1 kinase activity prevents atherosclerotic progression and inflammation in HFD‐fed ApoE−/− mice. A) Representative images of plaque lesion in aortic arches. B) Representative whole‐aorta Oil Red O staining. C) Representative images of Oil Red O staining of atherosclerotic lesion in aortic roots (Scale bar = 250 µm). D) Representative images of Masson's Trichrome staining in aortic roots (Scale bar = 250 µm). E) Representative images of Sirius Red staining in aortic roots (Scale bar = 50 µm). F) Representative immunofluorescence staining images of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar = 250 µm. G,H) Representative immunohistochemistry staining images of Ly6G G) and Ly6C H) in aortic roots (Scale bar = 25 µm). I) Scatter diagram of neutrophils and monocytes in plasma measured by an automated blood cell analyzer. SSC, side scatter; SFL, side fluorescence. J–M) Protein J,K) and mRNA L,M) levels of inflammatory cytokines TNF‐α and IL‐6 in serum or aortas. The values of mRNA levels were normalized to Rn18s (n = 6). Data were shown as mean ± SEM. J–M, one‐way ANOVA followed by Tukey's test.