Supplementary Figure 1

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Scarlett Gordon
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1 Supplementary Figure Bregma -1.6mm 3 : Bregma Ref) Bregma -.18mm Supplementary Figure 1 Schematic representation of the utilized brain slice for TTC staining and immunohistochemistry, which was located at -1.6 mm and -.18 mm in adult mice (Modified from the atlas of Franklin and Paxinos 38. Confirming H/I model by TTC staining, the brain tissues from the boxed ipsilateral penumbra (3) and contralateral area () were used for RT-PCR and Western blot analyses.

Supplementary Figure a 16 Iba-1 Iba-1 67 65 GAD 5

6mm) d e SSC SSC 11% % Iba-1 Iba-1 3% % 535bp 738bp

2 Supplementary Figure a 16 Iba-1 Iba GAD 5 GFAP GFAP 3 MAP 8 6 NeuN NeuN 5 Tubulin 5 Tubulin Tubulin Mouse#3 Mouse#1 Mouse# b Iba-1 Merge c Counts () () Counts () () Iba-1 GFAP (Bregma:-1.6mm) d e SSC SSC 11% % Iba-1 Iba-1 3% % 535bp 738bp 91bp 3bp 11bp GFAP TREM Iba-1 MAP NeuN nd Control Ab nd Control Ab 317bp 3

3 Supplementary Figure Expression levels of glial or neuronal markers in brain tissues from the H/I mice and primary culture cells. (a) Westernblotanalysiswasperformedonbrain tissues from the H/I mice using the indicated antibodies. (b) Immunohistochemistry was performed with (red) and Iba-1 antibodies (green) on coronal sections of the H/I mice. Scale bar: m. (c-d) Brain cells isolated from ischemic ipsilateral and contralateral hemispheres were stained with antibodies against Iba-1, GFAP, or secondary antibody (Molecular probe, #A11), and analyzed by flow cytometry. The data shown are representative of at least three independent experiments. (e) RT-PCR analysis was performed in primary cultured glial cells and neuronal cells using the indicated primers. GFAP forward: 5`-AAG CTC CAA GAT GAA ACC AAC CTG A-3`, reverse: 5`-CCA CGA TGT TCC TCT TGA GGT GG-3`; and TREM forward: 5`-ATG GGA CCT CTC CAC CAG TT, reverse: 5`-TCA CGT ACC TCC GGG TCC A; and Iba-1 forward: 5`-GGA TTT GCA GGG AGG AAA AG-3`, reverse: 5`-TGG GAT CAT CGA GGA ATT G-3`; and MAP forward: 5`-TTG GCT CAC TTG ACA ATG CTC ACC-3`, reverse: 5`-AAT ATG ACA CCT GCT CAG AGC CCA-3`; and NeuN forward: 5`-CCA GGC ACT GAG GCC AGC ACACAG C-3`, reverse: 5`-CTC CGT GGG GTC GGA AGG GTG G-3`.

4 Supplementary Figure 3 Relative Luciferase Activity (relative to normoxic PGL3 basic control ) 1 3 PGL3 Basic Luc p(1517)luc (-1517~+1) muthre A A B C B C Luc Luc P=.7 P=. muthre B muthre C C Luc Luc :% O :1% O HRE A B C Supplementary Figure 3 expression is closely linked with HIF-1 Three putative HRE sites within p(1517)luc were targeted for mutagenesis. The mutations of base pairs in each of the HREs were made using a site-directed mutagenesis kit, and confirmed using DNA sequencing. The regions of mutated HREs are indicated at the bottom. Mouse primary mixed glial cells were transfected with the luciferase reporter constructs or PGL3-basic control reporter, and incubated under hypoxic or normoxic conditions for h. Relative promoter activity is expressed as the ratio of luciferase activity/β-galactosidase activity. 5

5 Supplementary Figure a Day 1 R C R:rostral C:caudal 6

Supplementary Figure (continued) b Day 1 Day 3 Day 5 Day 7 c R Day 1 C ADC map (A) (B) % of edema: lateral volume (B)-lateral volume (A) 1 lateral

Representative images of Coronal MRI (T weighted image) brain slices after h from H/I mice treated with 1 g of (n=)oranti-(n=).

(c) Representative images of ADC maps brain slices after h from H/I mice treated with 1 g of (n=)oranti- (n=).

6 Supplementary Figure (continued) b Day 1 Day 3 Day 5 Day 7 c R Day 1 C ADC map (A) (B) % of edema: lateral volume (B)-lateral volume (A) 1 lateral volume (A) Supplementary Figure A blocking antibody significantly reduces brain injury and edema formation after hypoxia-ischemia (a) Representative images of Coronal MRI (T weighted image) brain slices after h from H/I mice treated with 1 g of (n=)oranti-(n=). (b) Representative images of Coronal MRI (T weighted image) brain slices after indicated time from H/I mice treated with 1 g of (n=) or anti- (n=). (c) Representative images of ADC maps brain slices after h from H/I mice treated with 1 g of (n=)oranti- (n=). Edema volume was quantified with Image J analyzer and were calculated with [(ipsilateral volume-contralateral volume)/contralateral volume] 1 and expressed as a percentage of the damaged ipsilateral hemisphere. 7

7 Supplementary Figure 5 a MPO Gr-1 Merge MPO + Gr-1 + cells/mm 8 6 P=.9 (Bregma:-.18mm) b c MPO 381 bp 317 bp Relative level of MPO transcript 8 P=.3 6 MPO 6 Tubulin 5 Relative level of MPO protein P=.9 Supplementary Figure 5 Neutrophil migration is increased in the ipsilateral cortex of H/I mice. (a) Antibodies against MPO and Gr-1 were used to perform immunohistochemistry on coronal sections of the H/I mice h after injury. Scale bar: m. The graph shows the mean number of MPO + Gr-1 + cells per mm (mean s.d. from three independent experiments). (b) RT-PCR analysis and (c) Western blot analysis were performed on brain tissues from H/I mice with MPO-specific primers and antibody, respectively. Relative levels of MPO transcripts and proteins are shown as graphs. The data shown are representative of at least three independent experiments (b, p=.3 compared with the contralateral control; c, p=.9 compared with the contralateral control). 8

8 Supplementary Figure 6 a Splenocytes 1% O or % O Splenocytes 1% O or % O h Primary glial cells FACS analysis of floating cells in lower chamber % of Gr-1 high CD11b high cells Glia P=.16 : % O : 1% O Murine h embryonic fibroblast (MEF) FACS analysis of floating cells in lower chamber % of Gr-1 high CD11b high cells P=.6 P=.7 : % O : 1% O MEF (1X1 5 cells) MEF 1X1 5X1 1X1 5 (cells) b Splenocytes 1% O or % O Splenocytes 1% O or % O h Primary glial cells FACS analysis of floating cells in lower chamber % of Gr-1 high CD11b high cells P=.8 1 (O, %) % of Gr-1 high CD11b high cells h FACS analysis of floating cells in lower chamber P=.9 1 (O, %) 9

9 Supplementary Figure 6 (continued) c 8 wk old C57BL/6 Sorting for BM-derived neutrophils Sorted Neutrophils Gr-1 96% 1-day-old mouse pup Neutrophils h CD11b 1% O Glial cells Analysis of floating cells in lower chamber Supplementary Figure 6 The migration of neutrophils is increased in the presence of glial cells under hypoxic conditions. (a) Splenocytes were isolated from C57BL/6 mice, and incubated in a transwell system with primary glial cells or MEF cells (C57BL/6) under hypoxic or normoxic conditions for h. The cells were analyzed by flow cytometry using antibodies against CD11b and Gr-1. The percentage of Gr-1 high CD11b high cells is expressed as the mean s.d. from three independent experiments. (b) Splenocytes (5 1 5 ) were isolated from C57BL/6 mice, and incubated in a transwell system with or without 1 5 primary cultured glial cells under hypoxic or normoxic conditions for h. The percentage of Gr-1 high CD11b high cells is expressed as the mean ± s.d. from three independent experiments. (c) Schematic illustration of our transwell system-based neutrophil transmigration assay. 1

Supplementary Figure 7 DAPI MPO Neutrophil Merge (Bregma:-.18mm) MPO + Neutrophil + cells/mm 15 1 9 6 3 P=.

10 Supplementary Figure 7 DAPI MPO Neutrophil Merge (Bregma:-.18mm) MPO + Neutrophil + cells/mm P=.16 Supplementary Figure 7 The migration of neutrophils into ischemic ipsilateral regions is decreased in -blocking antibody-treated H/I mice. Representative confocal microscopic images of coronal sections from control - or blocking antibody-treated H/I mice. Scale bar: 5 m. 11

11 Supplementary Figure 8 a.7 - b Cortex ( h) Bregma H&E MPO Gr-1 Merge MPO Gr-1 Merge.7-1

12 Supplementary Figure 8 (continued) c Striatum ( h) MPO Gr-1 Merge MPO Gr-1 Merge cortex 1 striatum Bregma.7 1 Cortex ( h) MPO Gr-1 Merge MPO Gr-1 Merge 1 1 Bregma -. 13

13 Supplementary Figure 8 (continued) d Penumbra (Bregma.7) MPO Gr-1 Merge MPO Gr-1 Merge 1 day 3 days 5 days 7 days Supplementary Figure 8 The -blocking antibody reduces neutrophil migration after hypoxia-ischemia. (a) Schematic representation of the utilized brain slice for immunohistochemistry, which was located at bregma to - mm in adult mice (Modified from the atlas of Franklin and Paxinos 38 ). (b-d) Immunohistochemistry was performed on coronal sections from control - or -blocking-antibody-treated H/I mice at the indicated times after injury using antibodies against MPO and Gr-1. Scale bar: μm. 1

14 Supplementary Figure 9 a Relative HIF-1α mrna (%) P= HIF-1 +f/+f LysM-Hif-1 -/- b HIF-1 +f/+f LysM-Hif-1 -/- IL-1β 71 bp CXCL1 185 bp 317 bp Relative levels of IL-1 6 P=.19 contra Relative levels of CXCL1 HIF-1 +f/+f LysM-Hif-1 -/- HIF-1 +f/+f LysM-Hif-1 -/- 6 P=.6 Supplementary Figure 9 Similar events are observed in the primary glial cells and cortex tissues from LysM-Hif-1 -/- mice. (a) Real time quantitative RT-PCR analysis was performed in primary microglia cultured from HIF-1 +f/+f or LysM-Hif-1 -/- mice (n=6) using HIF-1 primer. (b) The transcript levels of IL-1 and CXCL1 were examined in brain tissues from the contralateral cortex and ischemic ipsilateral cortex of HIF-1 +f/+f or LysM-Hif-1 -/- mouse h after hypoxia and ischemic injury (n=3). Data shown are presented as the mean s.d. 15

15 Supplementary Figure 1 a Counts CD11b high CD5 low GFP low cells CD11b high CD5 low GFP high cells Relative MFI levels () P=. GFP low GFP high CD11b high CD5 low cells Counts GFAP high GFP low cells GFAP high GFP high cells Relative MFI levels () 3 1 P=.7 GFP low GFP high GFAP high cells b Coordinates from Bregma 1: AP +1, ML ±1.5, DV -3 : AP, ML ±1.5, DV -3 3: AP -1, ML ±1.5, DV -3 : AP -, ML ±1.5, DV -3 6 c Mice Lentivirus Number with the indicated score 1 3 n Mean Score (± s.d.) LysM- LV-GFP HIf-1α -/- LV-TIM3-GFP * Supplementary Figure 1 (a) LV-TIM3-GFP or LV-GFP was injected using an stereotaxic instrument. Five days after intracranial injection of lentiviral vectors, glial cells from the injected region were subjected to FACS analysis using antibody against (ebioscience, RMT-3-3). The graph shows the results from three mice. (b) Schematic drawing of the injection sites. (c) Neurological scoring was determined after h of H/I in mice LV-GFP (n=6) or LV-TIM3-GFP (n=6).

1b (C) (I) (C) (I) 13 (C) (I) 3 6 (33) 55 Tubulin (5) 95 7 HIF1α (1) 56 Tubulin 3 (5) b Blots and Gels;

16 Supplementary Figure 11 a Blots and Gels; Figure 1 Fig.1a 1bp (C) (I) (C) (I) (3bp) 1bp HIF1α (9bp) bp (C) (I) (317bp) Fig.1b (C) (I) (C) (I) 13 (C) (I) 3 6 (33) 55 Tubulin (5) 95 7 HIF1α (1) 56 Tubulin 3 (5) b Blots and Gels; Figure Fig.c Fig.d 1 (O,%) 1 (O,%) Fig.e IP: IP:HIF1α 1 1 (O,%) 1bp (3bp) (3bp) (6bp) 7bp 6bp IP: IP:HIF1α 1 (O,%) 1 (O,%) 1 1 (O,%) 1bp (317bp) (317bp) 6bp (6bp) Primary glial cell Neuron 17

17 Supplementary Figure 11 (continued) Fig.g HIF1 F/F HIF1 / Fig.h HIF1 F/F HIF1 / 1 1 (O,%) 1 1 (O,%) (3bp) 3 6 (33) 1bp HIF1 F/F HIF1 / HIF1 F/F HIF1 / 1 1 (O,%) 1bp 1 1 (O,%) HIF1α (9bp) 55 3 Tubulin (5) HIF1 F/F HIF1 / 1 1 (O,%) (317bp) 13 HIF1 F/F HIF1 / 1 1 (O,%) HIF1 (1) 95 HIF1 F/F HIF1 / 1 1 (O,%) Tubulin 56 (5) c Blots and Gels; Figure 3f Full length PARP (116) 55 3 Tubulin (5) 18

18 Supplementary Figure 11 (continued) d Blots and Gels; Figure Fig.a 7bp 6bp bp bp MPO (381bp) (317bp) Fig.b Tubulin (5) MPO (55~6) e Blots and Gels; Figure 5 Fig.5c Fig.5e 1 1 (O,%) 8bp 6bp bp IL-1β (71bp) bp IL-1β (71bp) 1 1 (O,%) 1bp CXCL1 (185bp) CXCL1 (185bp) 1 1 (O,%) bp (317bp) (317bp) 19

7a Microglia HIF-1 +f/+f LysM-Hif-1 -/- Microglia HIF-1 +f/+f LysM-Hif-1 -/- #1 # #1 # #1 # #1 # bp 1bp HIF1 (16bp) 1bp GAPDH (13bp) Fig.

19 Supplementary Figure 11 (continued) f Blots and Gels; Figure 6c HIF-1 +f/+f LysM-Hif-1 -/- HIF-1 +f/+f LysM-Hif-1 -/- HIF-1 +f/+f LysM-Hif-1 -/- 1 1 (O,%) 1 1 (O,%) 1 1 (O,%) bp bp bp CXCL1(185bp) IL-1β(71bp) (317bp) g Blots and Gels; Figure 7 Fig.7a Microglia HIF-1 +f/+f LysM-Hif-1 -/- Microglia HIF-1 +f/+f LysM-Hif-1 -/- #1 # #1 # #1 # #1 # bp 1bp HIF1 (16bp) 1bp GAPDH (13bp) Fig.7b HIF-1 +f/+f LysM-Hif-1 -/- HIF-1 +f/+f LysM-Hif-1 -/- bp (3bp) bp (317bp) Supplementary Figure 11 Full scans of blots and gels for Figure 1 (a), Figure (b), Figure 3 (c), Figure (d), Figure 5(e). Figure 6 (f), and Figure 7 (g).

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed.

Supplementary Table 3. 3 UTR primer sequences. Primer sequences used to amplify and clone the 3 UTR of each indicated gene are listed. Supplemental Figure 1. DLKI-DIO3 mirna/mrna complementarity. Complementarity between the indicated DLK1-DIO3 cluster mirnas and the UTR of SOX2, SOX9, HIF1A, ZEB1, ZEB2, STAT3 and CDH1with mirsvr and PhastCons