Number of animals used in each experiment and figure

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Supplemental Table: Number of animals used in each experiment and figure

Figure/Experiment Figure part Groups Group size No. of mice Figure 1

IHC 1C 2 3 6

Figure 2

Behavioral test 2A-C 3 23; 7; 5 35

Figure 3

PWL test 3A 2 16 32

PWL test 3B 3 7; 8; 7 22

PWF test 3C 2 17; 16 33

PWF test 3D 2 17; 16 33

Figure 4

IHC 4B 2 3 6

PWF test 4C, D 2 11; 23 34

PWF test 4E 2 11 22

Figure 5

In vivo GCaMP imaging 5B-E 2 5; 5 10

Figure 6

CPP 6A, B 2 10 20

CPP 6C, D 2 12 24

CPP 6E, F 2 10 20

Figure 7

Western blotting 7A-I 2 4; 7 11

Figure 8

Western blotting 8A, B 2 4; 7 11

Supplemental figure 1

Open field test S1A-D 2 19; 9 28

PWF test S2A-B 2 8; 8 16

Open field test S3A-F 3 6; 5; 6 17

Antibody test S4B 2 2; 1 3

Full blot image S5 0 0 0

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Supplemental Figure 1. Exploratory activity of wild-type mice at 6 weeks after T10 spinal cord injury (SCI). (A) Example traces of wild-type mouse exploratory activity in the open field test (10-minute duration) at 6 weeks after T10 SCI or sham surgery (skin incision). Three different zones (center, internal periphery and border periphery) are indicated. The total distance traveled (B), number of entries into each zone (C), and time spent in each zone (D) were comparable between SCI (n=19) and sham-operated (n=9) groups. Student’s t-test. Data are presented as mean + SD.

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Supplemental Figure 2. Systemic administration of dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA) did not inhibit mechanical hypersensitivity in wild-type mice with spinal cord injury (SCI). Subcutaneous injection of DALDA (1 mg/kg, s.c., n=8) did not change paw withdrawal frequency to low-force (0.07 g, A) or high-force (0.4 g, B) mechanical stimulation in wild-type mice at 6 weeks after SCI, as compared to that of mice treated with saline (n=8). Two- way mixed model ANOVA. Data are expressed as mean + SD.

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Supplemental Figure 3. Effects of systemic dermorphin [D-Arg2, Lys4] (1-4) amide (DALDA) administration on exploratory activity of wild-type mice after spinal cord injury (SCI). (A-C) Example traces show exploratory activity of SCI mice in the open field test (10- minute duration) before and 45 minutes after subcutaneous injection of saline (A, n=6), 1 mg/kg DALDA (B, n=5), and 10 mg/kg DALDA (C, n=6). The area between the outer black box and the inner blue box is the border periphery, the area between the blue box and the pink box is the internal periphery, and the pink box constitutes the center. (D) The total distance traveled, (E) the time spent in the center, and (F) the number of entries into the center before and after each drug treatment. Two-way mixed model ANOVA. Data are expressed as mean + SD.

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Supplemental Figure 4. Antibody specificity test. (A) Expression of mu-opioid receptor (MOR) protein in HEK293 cells transfected with or without MOR plasmids. (B) p-MOR-Ser³⁷⁵ expression in the lumbar spinal cord of global MOR knockout (MOR KO) mice (B6.129S2- Oprm1^tm1Kff/J, Jackson Laboratory) was much weaker than that in wild-type mice (WT).

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control.

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Supplemental Figure 5. Full Western blot images. (A-F) Western blots of MOR and p-MOR- Ser³⁷⁵ expression in the spinal cord above (A, D), at (B, E), and below (C, F) the level of injury in wild-type mice at 6 weeks after SCI (S, n=7) or sham surgery (control, C, n=4). (G, H) Full blots of MOR expression in the sciatic nerve (G) and DRG tissues (H) of mice after SCI (S, n=7) or sham surgery (control, C, n=4).