Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 1. Nuclear-localized FOXO1 is detected in angiogenic fronts which is essential for vessel sprouting (A) Angiogenic proceeding postnatal day-5 mice retina was dissected and immunostained with CD31 for vessel imaging (left). Retinas are divided into three areas; tip, vascular front, and vascular plexus, which are enlarged and assessed for the immunostaining using antibodies for FOXO1 (red), CD31 (white), and ESM1 (green). ESM1 is used as the tip marker. Data are representative of three independent experiments. Bar: 50 mm. (B) HUVECs formed spheroids and sprouted in the three-dimensional culture. A bright field differential interference contrast (DIC) image was shown on the left. Immunostaining with anti-FOXO1 antibody was shown in green color (middle). Arrows indicated the cells obtaining nuclear-localized FOXO1. Merged images and the high-power view of spout-front (tip-like) regions, representative in a-c, were shown on the right. Bar: 50 mm. (C) HUVECs spheroids were subjected to sprouting assay for 2 days in the presence of siRNA for control or FOXO1. Bar: 50 mm. The right graph indicates the number of sprouts from at least 5 independent experiments. Data are shown as mean G S.D. Black dot represents each individual value. *p < 0.005 compared with si-Control. See also Figures S1–S3.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Imaging, Immunostaining, Marker, Control

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 2. Genome-wide RNA-seq screening of FOXO1 regulated genes in the endothelium (A) MA plot of average gene expression (log2, X axis (A)) and the mean of log2-fold change (Y axis (M)) of si-FOXO1-mediated up or downregulated genes; these were compared with si-Control HUVECs treated with VEGF for 18 h. Red and blue dots indicate genes with substantial change in expression with M > 0.5 and A > 2.5. (B) MA plot of average gene expression (log2, X axis (A)) and the mean of log2-fold change (Y axis (M)) of Ad-FOXO1-mediated up or downregulated genes compared with that in Ad-Control treated HUVECs. Green and orange dots indicate genes with significantly altered expression with M > 0.5 and A > 3.0. (C) Venn diagram of overlapping si-FOXO1-, or Ad-FOXO1-mediated up or downregulated genes and genes responsive to 18-h VEGF treatment. Each number indicates gene volume. Yellow indicates the common responsive gene number. (D) Clustering and heatmap representation of the 44 common FOXO1-regulated genes from D. The color intensity indicates the log2-fold expression levels: yellow-higher and blue-lower, relative to the median in black. The gene name based on the reference sequence is shown on the right. (E) FOXO1-mediated Tip cell enrichment from Gene set enrichment analysis (GSEA). The normalized enrichment score (NES), P and q-values for the datasets from VEGF 18h vs. 0h (left), si-FOXO1 vs. si-control with 18h VEGF treatment (right), and Ad-FOXO1 vs. Ad-Control (bottom). See also Figure S4 and Tables S1, S2, S3, and S4.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Genome Wide, RNA Sequencing, Gene Expression, Control, Expressing, Sequencing

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 4. Genome-wide FOXO1-ChIP-seqs analysis in VEGF-treated endothelium (A) Correlation mapping with spearman score of 1st and 2nd FOXO1-ChIP-seqs. (B) Pie-chart of FOXO1-binding genome region in HUVECs using cis-regulatory element annotation system. (C) ChIP-qPCR validation of ESM1 loci from VEGF-treated HUVECs normalized to the input using the FOXO1-specific antibody and the control IgG. (D) Venn diagram with overlapping si-FOXO1- or Ad-FOXO1-mediated up or downregulated genes and the genes involved in the enrichment regions from FOXO1-ChIP-seq. Each number indicates the gene volume. Purple indicates the common responsive gene number. (E) FOXO1-ChIP-seq enrichment values are quantified with each tip vs. stalk mark categorized gene loci derived from the ref.32 N.D.: not detected.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Genome Wide, Binding Assay, ChIP-qPCR, Biomarker Discovery, Control, ChIP-sequencing, Derivative Assay

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 5. FOXO1 binds to both closed and open chromatin regions as a pioneer factor in the VEGF treated endothelium (A) FOXO1 enrichment patterns are compared with those of H3K27me3, H3K4me3, and H3K27ac histone codes. The FOXO1 enrichment around the peaks of each histone code (G5 kb) in HUVECs. (B) Integrated genome view of HOXA11 with 13 genome regions. Histone code information of H3K27ac (blue), H3K4me3 (black), and H3K27me3 (purple) from cells treated with VEGF for 0 h and 1 h, with adjusted peak levels shown at the upper left. Input indicates the negative control for FOXO1-ChIP-seq. (C) Integrated genome view of the indicated tip cell enriched gene loci. Blue and red broken rectangles indicate FOXO1 bound at the H3K27me3 and H3K27ac enriched areas, respectively. FOXO1 enrichment on the CXCR4 genome locus was derived from single ChIP-seq data because of the failure to get profound FOXO1 enrichment on the locus, using MACS peak call with another ChIP-seq. See also Figure S7.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Negative Control, ChIP-sequencing, Derivative Assay

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 6. FOXO1 expressed in ECs preferentially bind to EC-selective expressed genes (A) Venn diagram depicting the overlap of FOXO1 binding genes between HUVECs and B cells. (B) MEME co-enrichment analysis of FOXO1 ChIP-seq results from HUVECs (left) and B cells (right). The position-weight matrix indicates enriched sequences. The p value indicates the probability that the de novo enriched sequences obtained from ChIP-seq matched to the known consensus motifs by chance. (C) HUVEC- or B cell-specific and common enriched gene sets are subjected to Gene Ontology (GO) analysis. In each case, up to three GO terms with the highest significance and the corresponding p values are shown in the graph. (D) FOXO1 enrichment patterns and the scores in HUVECs (left) and B cells (right) around FOXO1-bound regions (G5 kb) in each cell type are shown. Heatmap density around the same genome loci is shown in the lower panel. (E) Integrated genome view of the representative HUVEC- or B-cell specific and common FOXO1 binding patterns. H3K4me3 and H3K27ac ChIP-seqs are shown as the proximal active promoter and enhancer mark, respectively. Input indicates the negative control for FOXO1-ChIP-seq from each cell. See Also Figure S8 and Tables S8 and S9.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Binding Assay, ChIP-sequencing, Negative Control

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells.

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet: Figure 7. FOXO1-bound enhancers played cell-type specific promoter activation in the endothelium (A) Schematic representation of ESM1 and ANGPT2 genome locus and the construction for luciferase reporter analysis with each FOXO1-bindable promoter and promoter+H3K27ac-positive enhancer. (B) ESM1 and ANGPT2 promoter activities in comparison with that of promoter less pGL4.10 in neuronal SH-SY5Y cells or HUVECs. The results show the mean G S.D. of luciferase right units (RLU) obtained in triplicate from three independent experiments. *p < 0.01 compared with the activity from SH-SY5Y. (C) ESM1 and ANGPT2 enhancer activities relative to each promoter in HUVECs. The results show the mean G S.D. of RLU obtained in triplicate from three independent experiments. *p < 0.01 and **p < 0.005 compared with each promoter alone.

Article Snippet: Antibodies FOXO1 Cell signaling technology Cat# 2880; RRID: AB_2106495 FITC-conjugated CD31 BD Pharmingen Cat# 555445; RRID: AB_395838 CD31 BD Pharmingen Cat#550274; RRID: AB_393571 ESM-1 R&D systems Cat# AF1999; RRID: AB_2101810 FOXO1 Abcam Cat#ab39670; RRID: AB_732421 Ser256 phosphorylated FOXO Cell Signaling Technology Cat#9461; RRID: AB_329831 b-actin Sigma-Aldrich Cat# A1978; RRID: AB_476692 Lamin A Santa Cruz Biotechnology Cat#sc-20680; RRID: AB_648168 b-tubulin WAKO Cat#014-25041; RRID: AB_2650453 HRP-conjugated anti-rabbit IgG Sigma-Aldrich Cat#A9169; RRID: AB_258434 HRP-conjugated anti-mouse IgG Sigma-Aldrich Cat#A9044; RRID: AB_258431 Phalloidin iFluor 647 Conjugate Cayman Cat#20555, RRID: AB_2620155 Alexa Fluor 488-conjugated anti-FITC Invitrogen Cat#A11096; RRID: AB_221588 Goat anti-Rabbit IgG, Alexa Fluor 594 Invitrogen Cat#A11037; RRID: AB_2534095 Donkey anti-Rat IgG, Alexa Fluor 488 Invitrogen Cat#A21208; RRID: AB_2535794 Donkey anti-Goat IgG, Alexa Fluor 647 Invitrogen Cat#A21447; RRID: AB_10925072 Bacterial and virus strains T24A/S256A/S319A FOXO1-IRES-EGFP adenovirus This paper N/A EGFP adenovirus This paper N/A ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus VectorBuilder VB230331 ANGPT2 enhancer region-targeting CRISPRCas9 & gRNAs #2 (#2-5’; 5’-AGCTGGAG ATGTGCCGCCAA-3’, #2-3’; 5’-CCGGCG GGCGGCTTCACGAC -3’) lentivirus VectorBuilder VB230331 EGFP control lentivirus VectorBuilder VB010000 Chemicals, peptides, and recombinant proteins EGM -2 bullet kit LONZA CC-3162 Dulbecco’s modified Eagle’s medium (DMEM) WAKO 041-30081 FBS Sigma-Aldrich 172012 Opti-MEM I Reduced Serum Medium gibco 31985-062 Hank’s buffered saline (HBSS) Nacalai 17459-55 Human VEGF-A WAKO 229-01313 Human TNF-a Peprotech 300-01A LB100 Selleck S7537 LY294002 Calbiochem 440202 SP600125 WAKO 197-16591 XMU-MP-1 MedChemExpress HY-100526 Everolimus WAKO 058-00456 (Continued on next page) iScience 27, 109161, March 15, 2024 15

Techniques: Activation Assay, Luciferase, Comparison, Activity Assay