Fig 2: Peripheral nerve injury-induced increased expression of ETS1 protein in mouse L3/4 injured DRGs. (a–c) CCI of unilateral sciatic nerve caused hypersensitivity to heat (a) and mechanical (b, c) stimuli. **p < 0.01, ***p < 0.001 versus sham; n = 8. Data were analyzed by two-way repeated-measures ANOVA followed by post hoc Tukey test. (d) Expression of Ets1 mRNA in the ipsilateral L3/4 DRGs on days 0, 3, 7, and 14 after CCI-induced chronic neuropathic pain. No significance versus day 0; n = 6. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (e) ETS1 protein expression in the ipsilateral L3/L4 DRGs on days 0, 3, 7, and 14 after CCI or sham surgery; n = 6 mice per time point. *p < 0.05, **p < 0.01 versus day 0. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (f) ETS1 protein expression in the ipsilateral L3/L4 DRGs on days 0, 3, 7, and 14 after spared nerve injury (SNI) or sham surgery; n = 6 mice per time point. **p < 0.01 versus day 0. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (g) ETS1 expression in the ipsilateral L3/L4 DRGs at 0 d, 2 h, 1 d, 3 d, 7 d, and 14 d after Freund’s adjuvant (CFA)-induced inflammation pain or sham surgery; n = 6 mice per time point. No significance versus day 0. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (h) Co-staining of ETS1 (red) and ß-tubulin III (a neuronal marker, green) immunofluorescence in the ipsilateral L3/4 DRGs on day 7 after CCI or sham surgery; n = 5. Scale bar, 30 µm. *p < 0.05 versus the corresponding sham group. Data were analyzed by Student’s t-test. PWF: Paw withdrawal frequency; PWL: Paw withdrawal latency.

Fig 3: Blocking DRG ETS1 upregulation alleviates neuropathic pain development and maintenance in mouse. (a) ETS1 protein levels in the ipsilateral L3/4 DRGs 7 days after CCI surgery in mice with microinjection of Ets1 siRNA (siRNA) and scrambled siRNA (Scr); n = 8 mice per group. **p < 0.01 versus the corresponding sham Scr + CCI Scr group. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (b–d) Effects of microinjection of Ets1 siRNA and scrambled siRNA into the ipsilateral L3/4 DRGs after CCI or sham surgery on ipsilateral paw withdrawal responses to mechanical (c, d) and thermal stimulation (b); n = 8 mice per group. ***p < 0.001 versus the corresponding sham Scr + CCI Scr group. ###p < 0.001 versus the corresponding CCI Scr + CCI siRNA group. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (e–g) Effects of microinjection of Ets1 siRNA and scrambled siRNA into the ipsilateral L3/4 DRGs after CCI or sham surgery on contralateral paw withdrawal responses to mechanical (f, g) and thermal stimulation (e); n = 8 mice per group. No significance versus the corresponding sham Scr + CCI Scr group. No significance versus the corresponding CCI Scr + CCI siRNA group. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (h) Locomotor function trial, including placing, grasping, and righting reflexes; n = 8 per group, five trials. No significance: Data were analyzed by one-way ANOVA followed by post hoc Tukey test.

Fig 4: Overexpression of ETS1 in DRG evoked neuropathic pain-like symptoms in naïve mice. (a) Overexpression of ETS1 with Lenti-Ets1 increased the expression of ETS1 in the ipsilateral DRGs of naïve mice as measured by western blotting. Gfp was used as a control for Lenti-Ets1. *p < 0.05 versus Gfp; n = 6. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (b–d) Effects of microinjection of Lenti-Ets1 into the ipsilateral L3/4 DRGs on the ipsilateral PWF in response to 0.07 g (c) and 0.4 g (d) von Frey filament stimuli and PWL in response to heat stimuli (b) at indicated days after microinjection; n = 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 versus Gfp. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (e–g) Effects of microinjection of Lenti-Ets1 into the ipsilateral L3/4 DRGs on the contralateral PWF in response to 0.07 g (f) and 0.4 g (g) von Frey filament stimuli and PWL in response to heat stimuli (e) at the indicated days after microinjection; n = 6 mouse per group. No significance versus Gfp. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (h) Locomotor function trial, including placing, grasping, and righting reflexes; n = 8 per group, five trials. No significance versus naïve: Data were analyzed by one-way ANOVA followed by post hoc Tukey test.

Fig 5: ETS1 positively regulates HDAC1 expression by targeting the promotor. (a) Schematic representation of ETS1 action on HDAC1 promotor region. The ETS1 binding region located in -272 ~ -257 of mouse HDAC1 promotor (transcription start site designated as +1) is marked in blue. (b–d) ChIP qPCR assay for ETS1 binding with HDAC1 promotor fragments, ipsilateral L3/4 DRGs on day 7 post-CCI or sham surgery (b); ipsilateral L3/4 DRGs on day 7 post-CCI or sham surgery with microinjection of Ets1 siRNA (siRNA) and scrambled siRNA (Scr) (c) and ipsilateral L3/4 DRGs microinjected with Lenti-Ets1 or Lenti-Gfp (d); PCR and DNA gel electrophoresis were performed to verify the binding results. Input, total purified fragments. M, ladder marker. IgG, negative control. n = 6. *p < 0.05, **p < 0.01 versus the sham group. Data were analyzed by Student’s t-test and one-way ANOVA followed by post hoc Tukey test. (e) Luciferase reporter analysis of ETS1 positive regulation of the transcription of HDAC1 with co-transfection of the reporter plasmid. *p < 0.05 versus the corresponding vectors; n = 3 repeats per treatment. The HDAC1 promotor bound by ETS1 was inserted into the luciferase promotor in the pGL6 vector. The constructed or empty pGL6 and ETS1 overexpression plasmid PCD-ETS1 were co-transfected into HEK293 T cells; H2O was used as control. Data were analyzed by one-way ANOVA followed by post hoc Tukey test. (f) HDAC1 protein expression in the ipsilateral L3/4 DRGs 7 days after CCI surgery in mice with microinjection of Ets1 siRNA (siRNA) and scrambled siRNA (Scr); n = 6 mice per group. *p < 0.05, **p < 0.01 versus sham group plus scrambled siRNA. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (g) HDAC1 protein expression in the ipsilateral L3/4 DRG 8 days after viral microinjection into the ipsilateral L3/4 DRGs in mice; n = 6 per group. **p < 0.01 versus the corresponding group, naïve as a control. Data were analyzed with one-way ANOVA followed by post hoc Tukey test. (h) Co-localization of ETS1 (green) with HDAC1 (red) in mice L3/4 DRGs neurons. Scale bar, 30 mm. (i–k) Effects of microinjection of Lenti-Ets1 mixed with siRNA Hdac1 (si-Hdac1) into the ipsilateral L3/4 DRGs on the ipsilateral PWF in response to 0.07 g (j) and 0.4 g (k) von Frey filament stimuli and PWL in response to heat stimuli (i) at the indicated days after microinjection and scrambled siRNA (Scr) as a control; n = 8 per group. ***p < 0.001 versus the corresponding group. Data were analyzed by two-way ANOVA followed by post hoc Tukey test. (l) Locomotor function trial, including placing, grasping, and righting reflexes; n = 8 per group, five trials. No significance versus naïve. Data were analyzed by one-way ANOVA followed by post hoc Tukey test.