Fig 2: Molecular similarities with CCMs and impact of Rbpj deletion in stroke. a Confocal images of cerebellar white matter (dashed outline) in P10 brains. Note dilated vessels (CD31+, red) and gliosis (GFAP+, green) in mutants. Scale bar, 50 µm. b RT-qPCR analysis in P10 sorted brain ECs. p-values, Brown–Forsythe and Welch one-way ANOVA with Tamhane’s T2 test (Krit1, Ccm2, and Pdcd10), and Kruskal–Wallis with Dunn’s test (Klf2 and Kl4), n = 4. c Confocal images showing Klf4 (white) and active integrin-ß1 (Itgß1(a), green) in blood vessels (GLUT1+, red) of P10 RbpjiPC cerebellar white matter (dashed outline). Scale bar, 50 µm. d, e Klf4 (d) and active integrin-ß1 (e) immunosignal fold change intensity in P10 vasculature. p-values, Student’s t-test, n = 5. f Confocal images showing perivascular PDGFRß (white) expression after stroke. Note abundance of PDGFRß+ GFP- cells (arrowheads, top panel) and morphological changes in GFP+ pericytes (arrows, bottom panel). Scale bar, 50 µm. g Representative T2-weight magnetic resonance imaging (MRI) 1 day after stroke. Scale bar, 2 mm. h MRI-based quantitation of space-occupying effect attributable to oedema (% HSE) and the lesion volume corrected (LVc, mm3) after stroke. FDR-adjusted p-values, one-way ANOVA with two stage linear step-up procedure of Benjamini, Krieger, and Yekutieli multiple comparison correction, n = 4. i Confocal images showing increased phSMAD3 (white; left panel) and infiltration of inflammatory cells (IB4+, blue; Iba1+, white; right panel) in RbpjiPC brains after stroke. GFAP staining (green, right panel) indicates boundary between infarct zone and penumbra (dashed line). Vessels are labelled by tdT (red). Scale bar, 100 µm (left) and 50 µm (right). j Confocal images showing blood vessel (tdT, red; VEGFR2, white) defects (arrowheads) in RbpjiPC mice 7 days post-stroke. Scale bar, 100 µm. k Gene expression fold change of pro-inflammatory chemokines in P10 RbpjiPC mural cells relative to controls (set as 1). p-adjusted values. n = 3. l RBPJ binding and H3K4me, H3K4me3 distribution at Ccl2 locus in pericytes. Black bar below RBPJ profiles indicates the position of peak intervals. Two replicates (Rep1 and Rep2) are shown. Error bars represent s.e.m. Source data are provided as a Source Data file

Fig 3: Blockade of the EGFR/MEK/ERK pathway increases keratinocyte expression of KLF4.(A) Quantitative PCR was performed to evaluate gene expression in RNA isolated from biopsies of 4 patients with acneiform eruption and 10 healthy control (HC) skin biopsies. Data represent mean ± SD. (B) PHKs were pre-exposed to the BRAF inhibitor vemurafenib (1 µg/mL) for 30 minutes and exposed to the MEK inhibitor trametinib (2 µg/mL) and C. acnes (MOI of 10) for 6 hours. Data represent mean ± SEM (n = 3). Data were analyzed with 2-tailed unpaired t test (A) or 1-way ANOVA followed by Tukey’s multiple-comparisons test (B). *P < 0.05; **P < 0.01; ***P < 0.001. (C) PHKs were exposed to erlotinib (1 µM) or trametinib (2 µg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and ß-actin. (D) ERK1 and ERK2 were silenced by siRNA in PHKs and cell lysates were analyzed by SDS-PAGE and immunoblotting with indicated antibodies. (E) HEK293T cells were transfected with FLAG-tagged KLF4 and Myc-tagged ERK1 and ERK2 for 24 hours. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. (F) HEK293T cells were transfected with FLAG-tagged KLF4, HA-tagged ubiquitin, and constitutively active ERK (CA-ERK) for 24 hours. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments.

Fig 4: The effects of Prmt1 inhibition and R396A mutant of Klf4 on the PrE commitment. (A) Scheme of the chimeric assay. (B) Chimeric assay of ES cells with (Fur+) or without (Fur–) treatment. The ES cell line was AB2.2 cells with GFP and td-Tomato. After Fur treatment for 24 h, cells were microinjected into blastocysts. Embryos were observed at E6.5. GFP (green) and Td-Tomato (red) signals were detected. Dashed lines mark epiblasts. (C) IF assays showed the effect of Fur on mouse blastocysts. Blastocysts were cultured with (Fur+) or without (Fur–) for 24 h and then immunostained with Gata6 (red) to indicate PrE and Nanog (green) to indicate EPI. (D) Distribution of two cell types in the ICM. (E) IF assays showed the impact of Fur on the expression of Gata6 (green) and Nanog (red) in E14 cells with XEN induction. Nuclei were visualized with DAPI staining (blue). Yellow circles indicate Gata6-positive cells. (F) Fur treatment induced the expression of Gata4 and Gata6 genes using RT-qPCR, and Nestin was used as a negative control. (G) Chimeric assay of ES cells of wild type Klf4 and mutant Klf4 R396A. The AB2.2 cells were microjnjected into blastocysts. Embryos were observed at E6.5. Dashed yellow lines marked epiblasts, the arrows indicated chimeric cells outside of epiblasts. (H) IF assays showed the impact of Klf4 R396A mutation in AB2.2 cells. Nuclei were visualized with DAPI staining (blue). Yellow circles indicate Gata6-positive cells. (I) RT-qPCR assay showed that XEN induction further increased the expression of Gata4 and gata6 genes in R396A mutation in AB2.2 ES cells. (J) Schematic depiction of Klf4 methylation by Prmt1 to regulate PrE gene expression via mSin3a/HDAC recruitment in mouse ESCs, which heterogeneously prevents a bias toward PrE commitment.

Fig 5: KLF4-bound regions carrying epigenetic enhancer marks are enriched in loci of some genes associated with neural differentiation. (A) Percentage of KLF4-bound sites in annotated TSS-linked regions (TSS ± 2 kb) and in bodies of genes significantly up- or down-regulated in neural progenitor cells (NPCs). (B) Percentage of KLF4-bound sites in TSS-linked regions (TSS ± 2 kb) and non-TSS regions in mESCs. (C) Percentage of marks of enhancers and active enhancers in regions within KLF4-bound TSS-linked regions (TSS ± 2 kb) and non-TSS regions in mESCs. (D) Percentage of marks of enhancers and poised enhancers in regions within KLF4-bound TSS-linked regions (TSS ± 2 kb) and non-TSS regions in mESCs. (E) KLF4-bound regions that carry epigenetic enhancer marks at Pax6, Sox4, Foxp4 and Satb2 gene loci.