Table S1. Statistical analysis results for each figure
Figures Statistic Method Factor 1 Factor 2 Factor 3 F（df）/P
Fig. 1A Three-way ANOVA
Ftime(3,176) = 31.58 P < 0.0001
Fmodel(1,176) = 320.3
P < 0.0001
Fside(1,176) = 208.3
P < 0.0001
Fmodel×side×time（3,176） = 27.03
P < 0.0001
Fig. 1B Three-way ANOVA
Ftime(3,176) = 26.32 P < 0.0001
Fmodel(1,176) = 260.7
P < 0.0001
Fside(1,176) = 333.4
P < 0.0001
Fmodel×side×time（3,176） = 22.07
P < 0.0001
Fig. 1C Three-way ANOVA
Ftime(3,176) = 37.83 P < 0.0001
Fmodel(1,176) = 505.5
P < 0.0001
Fside(1,176) = 425.0
P < 0.0001
Fmodel×side×time（3,176） = 50.23
P < 0.0001
Fig. 1D Two-way ANOVA
Ftime(3,88) = 267.2 P < 0.0001
Fmodel(1,88) = 1704 P < 0.0001
Fmodel×time（3,88）= 282.3 P < 0.0001
Fig. 1E Two-way ANOVA
Ftime(3,16) = 11.32 P = 0.0003
Fmodel(1,16) =
52.38 P < 0.0001
Fmodel×time（3,16）= 5.366 P = 0.0095
Fig. 1F Two-way ANOVA
Ftime(3,16) = 5.075 P = 0.0117
Fmodel(1,16) =
47.94 P < 0.0001
Fmodel×time（3,16）= 5.858 P = 0.0067
Fig. 1G for Con DRG
One-way ANOVA
F（3,8）= 0.2781 P = 0.8398
Fig. 1G for Ipsi SC
One-way ANOVA
F（3,8）= 1.734 P = 0.2371
Fig. 1H Two-way ANOVA
Ftime(3,16) = 9.658 P=0.0007
Fmodel(1,16) =
66.70 P < 0.0001
Fmodel×time（3,16）= 8.188 P = 0.0016
Fig. 1I for Con L4 DRG
One-way ANOVA
F（3,8）= 0.3013 P = 0.8238
Fig. 1I for Ipsi L3 DRG
One-way ANOVA
F（3,8）= 0.02533 P = 0.9941
Fig. 1I for Ipsi L4 SC
One-way ANOVA
F（3,8）= 0.05797 P = 0.9804
Fig. 2H Two-tailed unpaired Student’s t-test
t =5.535 P = 0.0002
Fig. 3A Two-way ANOVA
Ftreatment(1,8) = 15.95 P = 0.0040
Fmodel(1,8) = 13.52 P = 0.0063
Ftreatment×model（1,8）= 6.084 P = 0.0389
Fig. 3B Three-way ANOVA
Ftime(6,308) = 52.13 P < 0.0001
Fmodel(1,308) = 816.7
P < 0.0001
Ftreatment(1,308)
= 301.0 P < 0.0001
Ftime×treatment×model（6,308）= 23.82
P < 0.0001
Fig. 3C Three-way ANOVA
Ftime(6,308) = 37.34 P < 0.0001
Fmodel(1,308) = 462.9
Ftreatment(1,308)
= 141.2
Ftime×treatment×model（6,308）= 8.960
P < 0.0001 P < 0.0001 P < 0.0001
Fig. 3D Three-way ANOVA
Ftime(6,308) = 90.8 P < 0.0001
Fmodel(1,308) = 811.1
P < 0.0001
Ftreatment(1,308)
= 252.8 P < 0.0001
Ftime×treatment×model（6,308）=
27.79 P < 0.0001
Fig. 3E Three-way ANOVA
Ftime(6,308) = 234.1 P < 0.0001
Fmodel(1,308) = 2361
P < 0.0001
Ftreatment(1,308)
= 1672 P < 0.0001
Ftime×treatment×model（6,308）= 150.6
P < 0.0001
Fig. 3F Three-way ANOVA
Ftime(6,308) = 1.020 P =0.412
Fmodel(1,308) = 0.106
P = 0.745
Ftreatment(1,308)
= 0.01178 P = 0.9137
Ftime×treatment×model（6,308）=
0.5522 P = 0.7682
Fig. 3G Three-way ANOVA
Ftime(6,308) = 1.540 P =0.1645
Fmodel(1,308) = 3.795
P = 0.0523
Ftreatment(1,308)
= 0.7455 P = 0.3886
Ftime×treatment×model（6,308）= 0.9359
P = 0.4693
Fig. 3H Three-way ANOVA
Ftime(6,308) = 2.960 P =0.008
Fmodel(1,308) = 0.1289
P = 0.7198
Ftreatment(1,308)
= 0.0000 P ＞ 0.9999
Ftime×treatment×model（6,308）= 0.4007
P = 0.8784
Fig. 3I for p-ERK1
Two-way ANOVA
Ftreatment(1.8) = 220.1 P < 0.0001
Fmodel(1,8) = 197.7 P < 0.0001
Ftreatment×model（1,8）= 151.2 P < 0.0001
Fig. 3I for p-ERK2
Two-way ANOVA
Ftreatment(1.8) = 96.29 P < 0.0001
Fmodel(1,8) = 64.85 P < 0.0001
Ftreatment×model（1,8）= 76.44 P < 0.0001
Fig. 3I for GFAP
Two-way ANOVA
Ftreatment(1.8) = 21.92 P = 0.0016
Fmodel(1,8) = 22.30 P = 0.0015
Ftreatment×model（1,8）= 11.27 P = 0.01
Fig. 3I for ERK1
Two-way ANOVA
Ftreatment(1.8) = 0.5888 P = 0.4648
Fmodel(1,8) = 2.195 P = 0.1767
Ftreatment×model（1,8）= 0.03479 P = 0.8567
Fig. 3I for ERK2
Two-way ANOVA
Ftreatment(1.8) =0.1035 P = 0.7559
Fmodel(1,8) = 1.745 P = 0.2231
Ftreatment×model（1,8）= 0.03219 P = 0.8621
Fig. 4A Two-way ANOVA
Ftime(7,144) = 138.8 P < 0.0001
Ftreatment(1,144) = 136.9
P < 0.0001
Ftime×treatment（7,144）= 45.05 P < 0.0001
Fig. 4B Two-way ANOVA
Ftime(7,144) = 71.31 P < 0.0001
Ftreatment(1,144) = 108.4
P < 0.0001
Ftime×treatment（7,144）= 25.19 P < 0.0001
Fig. 4C Two-way ANOVA
Ftime(7,144) = 24.91 P < 0.0001
Ftreatment(1,144) = 23.07
P < 0.0001
Ftime×treatment（7,144）= 8.259 P < 0.0001
Fig. 4D Two-way ANOVA
Ftime(7,144) = 159.0 P < 0.0001
Ftreatment(1,144) = 308.7
P < 0.0001
Ftime×treatment（7,144）= 72.27 P < 0.0001
Fig. 4E Two-way ANOVA
Ftime(7,144) = 1.037 P = 0.4081
Ftreatment(1,144) = 0.000
P > 0.9999
Ftime×treatment（7,144）= 0.4977 P = 0.8350
Fig. 4F Two-way ANOVA
Ftime(7,144) = 2.136 P = 0.0435
Ftreatment(1,144) = 1.256
P = 0.2642
Ftime×treatment（7,144）= 0.3773 P = 0.9144
Fig. 4G Two-way ANOVA
Ftime(7,144) = 0.3057 P =0.9504
Ftreatment(1,144) = 0.04339
P = 0.8353
Ftime×treatment（7,144）= 0.1874 P = 0.9877
Fig. 4H One-way ANOVA
F（2,6）= 11.70 P = 0.0085
Fig. 4I for p-ERK1
One-way ANOVA
F（2,6）= 28.55 P = 0.0009
Fig. 4I for p-ERK2
One-way ANOVA
F（2,6）= 21.64 P = 0.0018
Fig. 4I for GFAP
One-way ANOVA
F（2,6）= 21.47 P = 0.0018
Fig. 4I for ERK1
One-way ANOVA
F（2,6）= 0.6037 P = 0.5769
Fig. 4I for ERK2
One-way ANOVA
F（2,6）= 4.890 P = 0.0550
Fig. 5A Three-way ANOVA
Ftime(6,308) = 4.978 P < 0.0001
Ftreatment(1,308) = 53.35
P < 0.0001
Fside(1,308) = 44.32
P < 0.0001
Ftreatment×side×time（6,308）= 6.296
P < 0.0001
Fig. 5B Three-way ANOVA
Ftime(6,308) = 6.454 P < 0.0001
Ftreatment(1,308) = 24.83
P < 0.0001
Fside(1,308) = 36.46
P < 0.0001
Ftreatment×side×time（6,308）= 5.074
P < 0.0001
Fig. 5C Three-way ANOVA
Ftime(6,308) = 5.797 P < 0.0001
Ftreatment(1,308) = 23.95
P < 0.0001
Fside(1,308) = 24.57
P < 0.0001
Ftreatment×side×time（6,308）= 5.869
P < 0.0001
Fig. 5D Two-way ANOVA
Ftime(6,154) = 43.13 P < 0.0001
Ftreatment(1,154) = 158.8
P < 0.0001
Ftreatment×time（6,154）= 38.70 P < 0.0001
Fig. 5E Three-way ANOVA
Fpaired(1,11) = 21.32 P = 0.0007
Ftreatment(1,11) = 0.4281
P = 0.5264
Ftime(1,11) = 0.4281 P = 0.5264
Fpaired×treatment×time（1,11）= 17.68
P = 0.0015
Fig. 5F Two-tailed unpaired Student’s t-test
t = 4.002 P = 0.0006
Fig. 5G Two-tailed unpaired Student’s t-test
t = 4.178 P = 0.0139
Fig. 5H for p-ERK1
Two-tailed unpaired Student’s t-test
t = 6.264 P = 0.0033
Fig. 5H for p-ERK2
Two-tailed unpaired Student’s t-test
t = 6.530 P = 0.0028
Fig. 5H for GFAP
Two-tailed unpaired
t = 4.628 P = 0.0098
Student’s t-test Fig. 5H for
ERK1
Two-tailed unpaired Student’s t-test
t = 0.2526 P = 0.8130
Fig. 5H for ERK2
Two-tailed unpaired Student’s t-test
t = 0.2458 P = 0.8179
Fig. 6A Two-way ANOVA
Ftreatment(1.8) = 33.52 P = 0.0004
Fmodel(1,8) = 37.68 P = 0.0003
Ftreatment×model（1,8）= 19.44 P = 0.0023
Fig. 6B One-way ANOVA
F（2,6）= 12.80 P = 0.0068
Fig. 6C Two-tailed unpaired Student’s t-test
t =15.13 P = 0.0001
Fig. 6D for ELF1
One-way ANOVA
F（3,8）= 23.28 P = 0.0003
Fig. 6D for MMP9
One-way ANOVA
F（3,8）= 9.269 P = 0.0056
Fig. 6E One-way ANOVA
F（3,8）= 289.8 P < 0.0001
Fig. 6F Two-tailed paired Student’s t-test
t =16.23 P = 0.0038
Fig. S1A Two-way ANOVA
Ftreatment(1,8) = 96.15 P = 0.0009
Fmodel(1,8) = 88.62 P < 0.0001
Ftreatment×model（1,8）= 26.00 P = 0.0009
Fig. S1B Three-way ANOVA
Ftime(6,140) = 28.79 P < 0.0001
Fmodel(1,140) = 304.5
P < 0.0001
Ftreatment(1,140)
= 147.7 P < 0.0001
Ftime×treatment×model（6,140）=
8.487 P < 0.0001
Fig. S1C Three-way ANOVA
Ftime(6,140) = 24.81 P < 0.0001
Fmodel(1,140) = 312.1
P < 0.0001
Ftreatment(1,140)
= 106.8 P < 0.0001
Ftime×treatment×model（6,140）=
9.056 P < 0.0001
Fig. 1D Three-way ANOVA
Ftime(6,140) = 122.1 P < 0.0001
Fmodel(1,140) = 2002
P < 0.0001
Ftreatment(1,140)
= 522.2 P < 0.0001
Ftime×treatment×model（6,140）=
40.36 P < 0.0001
Fig. S1E Three-way ANOVA
Ftime(6,140) = 119.1 P < 0.0001
Fmodel(1,140) = 2025
P < 0.0001
Ftreatment(1,140)
= 1195 P < 0.0001
Ftime×treatment×model（6,140）=
81.87 P < 0.0001
Fig. S1F Three-way ANOVA
Ftime(6,140) = 0.09887 P = 0.9964
Fmodel(1,140) = 0.3390
P = 0.5614
Ftreatment(1,140)
= 0.08475 P = 0.7714
Ftime×treatment×model（6,140）=
0.09887 P = 0.9964
Fig. S1G Three-way ANOVA
Ftime(6,140) = 0.7417 P = 0.6170
Fmodel(1,140) = 0.000
P ＞ 0.9999
Ftreatment(1,140)
= 0.1000 P = 0.7523
Ftime×treatment×model（6,140）=
0.4583 P = 0.8381
Fig. S1H Three-way Ftime(6,140) = 0.4805 Fmodel(1,140) = Ftreatment(1,140) Ftime×treatment×model（6,140）=
ANOVA P =0.8220 0.1533 P =0.6960
= 0.1911 P =0.6627
0.06646 P =0.9988
Fig. S1I for p-ERK1
Two-way ANOVA
Ftreatment(1.8) = 88.93 P < 0.0001
Fmodel(1,8) = 104.0 P < 0.0001
Ftreatment×model（1,8）= 75.0 P < 0.0001
Fig. S1I for p-ERK2
Two-way ANOVA
Ftreatment(1.8) = 290.1 P < 0.0001
Fmodel(1,8) = 234.1 P < 0.0001
Ftreatment×model（1,8）= 234.1 P < 0.0001
Fig. S1I for GFAP
Two-way ANOVA
Ftreatment(1.8) = 92.59 P < 0.0001
Fmodel(1,8) = 100.1 P < 0.0001
Ftreatment×model（1,8）= 65.33 P < 0.0001
Fig. S1I for ERK1
Two-way ANOVA
Ftreatment(1.8)=
1.479e-029 P ＞ 0.9999
Fmodel(1,8) =
0.3333 P = 0.5796
Ftreatment×model（1,8）= 0.3333 P = 0.5796
Fig. S1I for ERK2
Two-way ANOVA
Ftreatment(1.8)= 0.3077 P = 0.5943
Fmodel(1,8) = 1.231 P = 0.2995
Ftreatment×model（1,8）= 0.3077 P = 0.5943
Fig. S2A Three-way ANOVA
Ftime(6,140) = 14.96 P < 0.0001
Ftreatment(1,140) = 59.77
P < 0.0001
Fside(1,140) = 74.06
P < 0.0001
Ftreatment×side×time（6,140）= 10.92
P < 0.0001
Fig. S2B Three-way ANOVA
Ftime(6,140) = 17.89 P < 0.0001
Ftreatment(1,140 )=
129.5 P < 0.0001
Fside(1,140) = 118.7
P < 0.0001
Ftreatment×side×time（6,140）= 15.64
P < 0.0001
Fig. S2C Three-way ANOVA
Ftime(6,140) = 52.96 P < 0.0001
Ftreatment(1,140) = 205.9
P < 0.0001
Fside(1,140) = 276.0
P < 0.0001
Ftreatment×side×time（6,140）= 38.85
P < 0.0001
Fig. S2D Two-way ANOVA
Ftime(6,70) = 71.55 P < 0.0001
Ftreatment(1,70) = 388.2
P < 0.0001
Ftreatment×time（6,70）= 58.76 P < 0.0001
Fig. S2E Three-way ANOVA
Fpaired(1,5) = 0.1553 P= 0.7098
Ftreatment(1,5) = 6.667
P= 0.0493
Ftime(1,5) = 4.0 P = 0.1019
Fpaired×treatment×time（1,5） = 19.93
P = 0.0066
Fig. S2F Two-tailed unpaired Student’s t-test
t = 4.841 P = 0.0007
Fig. S2G Two-tailed unpaired Student’s t-test
t = 13.94 P = 0.0002
Fig. S2H for p-ERK1
Two-tailed unpaired Student’s t-test
t = 33.23 P < 0.0001
Fig. S2H for p-ERK2
Two-tailed unpaired Student’s t-test
t = 13.87 P = 0.0002
Fig. S2H for GFAP
Two-tailed unpaired Student’s t-test
t = 12.25 P = 0.0003
Fig. S2H for Two-tailed t = 0.5000
ERK1 unpaired Student’s t-test
P = 0.6433
Fig. S2H for ERK2
Two-tailed unpaired Student’s t-test
t = 2.111 P = 0.1024
-35 -1 3 5 7 10 14 6
8 10 12
14 Scr+CCI
-35 -1 3 5 7 10 14 6
8 10 12 14
-35 -1 3 5 7 10 14 20
40 60 80
-35 -1 3 5 7 10 14 0
20 40 60
-35 -1 3 5 7 10 14 5
10 15
20 Sh+CCI
-35 -1 3 5 7 10 14 20
40 60
80 Sh+Sham
-35 -1 3 5 7 10 14 0
20 40 60
80 Scr+Sham
ERK1 ERK2 pERK1 pERK2 GFAP 0
1 2 3 4 0
1 2 3
F
P W F (%) P W F (%) P W L ( s) P W L ( s)
Days after CCI or sham surgery
0.07g 0.4g Heat
** ** ** ** **
** ** **
** **
** ** ** ** **
AAV
AAV
AAV
Cold
** ** ** ** **
AAV
B C D E
P W F (%) P W F (%) P W L ( s)
Days after CCI or sham surgery
0.07g 0.4g Heat
AAV AAV
AAV
G H
R elativ e lev el **
##
H3 ELF1
Sham CCI CCI Sham
Scr Sh
A
Scr
Sham CCI CCI Sham
Sh Scr + Sham Scr + CCI
Sh + CCI Sh + Sham
I
R elativ e lev el
GFAP ERK1/2 p-ERK1/2
GAPDH
** ** **
## ## ##
Figure S1. Effect of DRG microinjection of AAV5-Elf1 shRNA on CCI-induced nociceptive hypersensitivities and dorsal horn central sensitization during the development period in
female mice. (A) Expression of ELF1 protein in the ipsilateral L3/4 DRGs on day 14 after CCI or sham surgery in mice microinjected with AAV5-Elf1 shRNA (Sh) or negative
control AAV5-scramble shRNA (Scr). n = 3 experimental repeats (6 mice) /group. Two-way ANOVA followed by post hoc Tukey test (Table S1). **P < 0.01 vs the scrambled shRNA
plus sham group. ##P < 0.01 vs the scrambled shRNA plus CCI group. (B-H) Effect of microinjection of AAV5-Elf1 shRNA (Sh) or negative control AAV5-scrambled shRNA (Scr)
into the ipsilateral L3/L4 DRGs on paw withdrawal frequencies (PWF) to 0.07 g (B, F) and 0.4 g (C, G) von Frey filament stimuli and paw withdrawal latency (PWL) to heat (D, H)
and cold (E) stimuli on the ipsilateral (B-E) and contralateral (F-H) sides at the different days as indicated after CCI or sham surgery. n = 6 mice/group. Three-way ANOVA with
repeated measures followed by post hoc Tukey test (Table S1). **P < 0.01 vs the scrambled shRNA plus CCI group at the corresponding time points. (I) Effect of microinjection of
AAV5-Elf1 shRNA (Sh) or negative control AAV5-scrambled shRNA (Scr) into the ipsilateral L3/L4 DRGS on the expression of p-ERK1/2, total ERK1/2 and GFAP in the ipsilateral
L3/L4 dorsal horn on day 14 after CCI or sham surgery. Left: representative Western immunoblots. Right: statistical summary of densitometric analysis. n = 3 mice/group. Two-way
ANOVA followed by post hoc Tukey test (Table S1). **P < 0.01 vs the corresponding scrambled shRNA plus sham group. ##P < 0.01 vs the corresponding scrambled shRNA plus
CCI group.
-100 0 100 200
0 200 400 600 800
BL 2 3 4 5 7 9
5 10 15
20 GFP Ipsi
BL 2 3 4 5 7 9
8 9 10 11 12
13 GFP Con
BL 2 3 4 5 7 9
20 40 60 80
100 ELF1 Con
BL 2 3 4 5 7 9
0 20 40
60 ELF1 Ipsi
ERK1 ERK2 pERK1 pERK2 GFAP 0
1 2 3
0 1 2 3 4
Weeks after AAV5 microinjection
A
T im e (s )
Pre Post Pre Post
GFP ELF1
Saline-paired Lidocaine-paired
E
Dif fer en ce s co re (s ) (P o st -P re
）
F
R elativ e L ev el
G H
GFP ELF1
R elativ e lev el
**
**
**
*
** ** ** ** ** **
**
** **
**
** **
P W F (%) P W F (%) P W L ( s) P W L ( s)
0.07g B 0.4g C Heat D Cold
** ** **
GFAP ERK1/2 p-ERK1/2
GAPDH
GFP ELF1
GFP ELF1 GFP ELF1
ELF1 H3
* **
Figure S2. Effect of DRG ELF1 overexpression on nociceptive thresholds and dorsal horn central sensitization in naïve female mice. (A-D) Effect of microinjection of AAV5- Elf1 (ELF1) or AAV5-Gfp (GFP) into the unilateral L3/4 DRGs on paw withdrawal frequencies (PWF) to 0.07 g (A) and 0.4 g (B) von Frey filament stimuli and paw
withdrawal latency (PWL) to heat (C) and cold stimuli (D) on the ipsilateral (Ipsi) and contralateral (Con) sides at the different weeks as indicated post-viral microinjection.
BL, baseline. n = 6 mice/group. Three-way ANOVA (A-C) or two-way ANOVA (D) with repeated measures followed by post hoc Tukey test (Table S1). *P < 0.05, **P < 0.01 vs the AAV5-Gfp group on the ipsilateral side at the corresponding time points. (E, F) Effect of microinjection of AAV5-Elf1 or AAV5-Gfp into the unilateral L3/4 DRGs on spontaneous ongoing pain as assessed by the CPP paradigm. E: Time spent in each chamber. F: Difference scores for chamber preference were calculated by subtracting preconditioning preference time from postconditioning time spent in the lidocaine-paired chamber. Pre, preconditioning. Post, post-conditioning. n = 6 mice/group. **P < 0.01 vs the corresponding preconditioning by three-way ANOVA followed by Tukey post hoc test (E; Table S1) or vs the AAV5-Gfp group by two-tailed unpaired Student’s t test (F;
Table S1). (G) Level of ELF1 protein in the ipsilateral L3/4 DRGs 9 weeks after microinjection of AAV5-Elf1 (ELF1) or control AAV5-Gfp (GFP). n = 6 mice/group. *P <
0.05 vs the AAV5-Gfp group by two-tailed unpaired Student’s t test (Table S1). (H) Effect of microinjection of AAV5-Elf1 (ELF1) or AAV5-Gfp (GFP) into the unilateral L3/4
DRGs on the levels of p-ERK1/2, total ERK1/2 and GFAP in the ipsilateral L3/4 dorsal horn 9 weeks after viral microinjection. Representative Western immunoblots (left) and
a summary of the densitometric analysis (right) are shown. n = 3 mice/group. **P < 0.01 vs the corresponding AAV5-Gfp group by two-tailed unpaired Student’s t test (Table
S1).