Figure 3: OCT1-triggered Dnmt3a gene transcription following peripheral nerve injury in rat injured DRG.
(a) The Dnmt3a promotor fragment immunoprecipitated by rabbit anti-OCT1 antibody in the ipsilateral L5 DRG on day 7 post-SNL or sham surgery. Input, total purified fragments. M, ladder marker. n=3 repeats. *P<0.05 versus the sham group by two-tailed unpaired Student’s t-test. (b) OCT1 expression in the ipsilateral L5 DRG after SNL or sham surgery. n=6 rats/time point/group. Two-way ANOVA followed by post hoc Tukey test, Ftime (2, 17)=33.0. **P<0.01 versus the corresponding control group (0 day). Full-length blots are presented in Supplementary Fig. 6. (c) Dnmt3a gene promotor activity in HEK-293 T cells transfected with the vectors and/or siRNAs as shown. Ctl: control empty pGL3-Basic. n=3 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, Fgroup (7, 23)=4,501.1. **P<0.01 versus the pGL3-Dnmt3a vector (Dnmt3a) alone. (d,e) Levels of OCT1 and DNMT3a proteins in PC12 cells transfected with the vectors as shown. Ctl: control pHpa-trs-GFP. Oct1: pHpa-trs-Oct1. siRNA: Oct1 siRNA. NC: negative control siRNA. N=5 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, Fgroup (5, 17)=13.8 for OCT1 and Fgroup (5, 17)=20.9 for DNMT3a. **P<0.01 versus control GFP-treated group. #P<0.05, ##P<0.01 versus the Oct1-treated group. Full-length blots are presented in Supplementary Fig. 6. (f) Levels of Oct1 mRNA and Dnmt3a mRNA in rat lumbar DRG cultured neurons transduced with AAV5-GFP (GFP) or AAV5-Oct1 (Oct1). n=3 repeats per treatment. One-way ANOVA followed by post hoc Tukey test, Fgroup (2, 8)=14.7 for Oct1 mRNA and Fgroup (2, 8)=10.2 for Dnmt3a mRNA. *P<0.05 or **P<0.01 versus the corresponding naive condition. (g) Co-expression of Oct1 mRNA with Dnmt3a mRNA in individual small, medium, and large neurons from the rat lumbar DRG. n=3 repeats.
