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. 2019 Aug 26;20(17):4177. doi: 10.3390/ijms20174177

Figure 4.

Figure 4

Neuropathic injury diminishes Hes1-repressed spinal mGluR5 transcription. (a,b) RT-PCR and western blot analyses (normalized to GAPDH) demonstrating that the mRNA and protein levels of mGluR5 in dorsal horn samples were increased by administering naïve animals (Naive) with Hes1 siRNA but not with missense siRNA (Hes1 siRNA and MS siRNA, respectively. 5 μg, 10 μL; it, daily for 4 days). IB, immunoblotting. mRNA, one-way ANOVA, post-hoc Tukey test, F (2,12) = 27.91, p < 0.0001. protein, one-way ANOVA, post-hoc Tukey test, F (2,15) = 34.80, p < 0.0001. ** p < 0.01 vs. Naïve. mRNA, n = 5. protein, n = 6. (c) ChIP-qPCR assay of dorsal horn samples demonstrating that administering naïve animals with Hes1 siRNA but not with missense siRNA (Hes1 siRNA and MS siRNA, respectively. 5 μg, 10 μL; it, daily for 4 days) decreased amounts of Hes1 antibody-precipitated exon 1a and exon 1b promoter fragments of mGluR5. ** p < 0.01 vs. Naïve. n = 5. Hes1, exon 1a, one-way ANOVA, post-hoc Tukey test, F (2,12) = 131.7, p < 0.0001. Hes1, exon 1b, one-way ANOVA, post-hoc Tukey test, F (2,12) = 22.70, p < 0.0001. (d,e) RT-PCR and western blot analyses of the ipsilateral dorsal horn samples dissected at day 21 after operation. When compared with the sham operation (Sham 21D), spinal nerve ligation (SNL 21D) increased mRNA and protein levels of mGluR5 that were both reversed by intrathecal administration to SNL animals with Hes1-encoding vector (SNL 21D + Hes1 LV) but not the control vector (SNL 21D + Control LV). mRNA, one-way ANOVA, post-hoc Tukey test, F(3,16) = 20.75, p < 0.0001. protein, one-way ANOVA, post-hoc Tukey test, F (3,20) = 34.70, p < 0.0001. ** p < 0.01 vs. Sham 21D. ## p < 0.01 vs. SNL 21D. mRNA, n = 5. protein, n = 6. (f) ChIP-qPCR assay of dorsal horn samples dissected at day 21 post operation. When compared with the sham operation, SNL decreased the amounts of Hes1 antibody-precipitated exon 1a and exon 1b promoter fragments of mGluR5; effects that were both reversed by transfecting animals with Hes1-encoding vector (SNL 21D + Hes1 LV) but not the control vector (SNL 21D + Control LV). Hes1, exon 1a, one-way ANOVA, post-hoc Tukey test, F (3,16) = 34.72, p < 0.0001. Hes1, exon 1b, one-way ANOVA, post-hoc Tukey test, F (3,16) = 45.74, p < 0.0001. ** p < 0.01 vs. Sham 21D. # p < 0.05, ## p < 0.01 vs. SNL 21D. n = 5.