Fig 2: Caspr1 depletion inhibits nuclear factor-?B (NF-?B) signaling pathway. (A) The cultured HBMECs with Caspr1 knockout were subjected to immunofluorescence with the primary antibody against the p65 subunit of NF-?B (red). DAPI (blue) was used for counterstaining. The fluorescence intensity of nuclear p65 divided by the p65 in whole cell (total p65) was quantified with ImageJ software. Images are from three independent experiments. Scale, 10 µm. **P < 0.01, Student’s t-test. (B) The nuclear proteins were extracted from cells, and the levels of the p65 were analyzed by Western blot. The nucleus marker, histone, served as loading control. The representative images were from three independent experiments. For quantification, the protein band intensities of the Western blot images were quantified with ImageJ software. Data are presented as mean ± SD for three independent experiments. *P < 0.05, Student’s t-test. (C) The activation of NF-?B upstream molecules including IKKß and I?Ba was analyzed by Western blot with antibodies recognizing phosphorylated IKKß (p-IKKß) and I?Ba (p-I?B). The representative images were from three independent experiments. For quantification of the phosphorylation levels, the protein band intensities of the phosphorylated protein were divided by that of the total protein. Data are normalized to Cas9 controls, which were defined as 1. Data are presented as mean ± SD for three independent experiments. *P < 0.05, Student’s t-test. (D,E) The HBMECs with Caspr1 knockout were stimulated with NF-?B agonist FSL1 and betalinic acid. (D) After 24 h, the total RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Then, real-time PCR was performed to detect the mRNA expression levels of ADAM9, with GAPDH as an internal control. Data are normalized to HBMECs, which was defined as 1. (E) The supernatant of HBMECs was collected; the secreted sAPPa was analyzed by ELISA. Values are mean ± SD from three independent experiment. ***P < 0.001, one-way ANOVA.

Fig 3: Restoration of Caspr1 expression rescues NF-κB deactivation, ADAM9 downregulation, and sAPPα reduction in Caspr1 knockout cells. (A) The HBMECs with Caspr1 knockout (Caspr1 KO) were infected with adenovirus coding the full-length Caspr1 (AdV-Caspr1), with the empty AdV used as a control. The infected Caspr1 KO was subsequently subjected to immunofluorescence with the primary antibody against p65 (red). DAPI (blue) was used for counterstaining. The fluorescence intensity of nuclear p65 was quantified with ImageJ software. Images are from three independent experiments. Scale, 10 μm. **P < 0.01, one-way ANOVA. (B,C) The HBMECs with Caspr1 KO were infected with adenovirus coding the full-length Caspr1 (AdV-Caspr1), with the empty AdV used as a control. After 48 h, the ADAM9 expression was examined by Western blot (B). At the same time, the concentration of sAPPα in the medium was measured by ELISA (C). All values are presented as mean ± SD for three independent experiments. ***P < 0.001, one-way ANOVA.

Fig 4: Caspr1 depletion downregulates ADAM9 mRNA levels. (A) The total RNA of HBMECs with Caspr1 knockout was extracted and reverse transcribed using Moloney murine leukemia virus (M-MLV) reverse transcriptase. Then, real-time polymerase chain reaction (PCR) was performed to detect the mRNA expression levels of ADAM9, ADAM10, and ADAM17, with GAPDH as an internal control. Data are normalized to HBMECs, which was defined as 1. Values are mean ± SD from three independent experiments. *P < 0.05, Student’s t-test. N.S., no statistical significance. (B) The truncated sequences of ADAM9 promoter were cloned into pGL3 vector encoding luciferase, respectively. The constructs were then transfected into 293T cells together with pRL-TK plasmids, followed by transfection with Caspr1 siRNA, with nonsilenceing siRNA as control. After 48 h, the cells were harvested and analyzed by luciferase reporter assay. *P < 0.05. **P < 0.01, ***P < 0.001, one-way analysis of variance (ANOVA). (C) The expressions of transcription factors including p65, Sp1, snail, and HIF-1a were detected by Western blot in HBMECs with Caspr1 knockout. GAPDH served as loading control. The representative images were from three independent experiments. For quantification, the protein band intensities of the Western blot images were quantified with ImageJ software. Data are normalized to Cas9 controls, which were defined as 1. Data are presented as mean ± SD for three independent experiments. (D) Caspr1-depleted HBMECs were subjected to ChIP assay using the NF-?B p65 antibody, whereas the isotype immunoglobulin G served as the control. The immunoprecipitated DNA fragments were amplified by real-time PCR using the primers flanking the promoter regions of ADAM9 genes. The expression levels were quantified, and the statistical analyses were performed. *P < 0.05, Student’s t-test.

Fig 5: Inhibition of NF-?B signaling attenuated pain behavior in the rats with bone cancer. (A) Implantation of tumor cells in the tibias significantly increased the phosphorylation of NF-?B subunit p65 (t = 2.93, P = 0.01), but not the total p65 (t = 0.54, P > 0.05), in the spinal dorsal horn of the rats (n = 7 rats in each group); (B) Blockade of NF-?B signaling by PDTC (1 µg, from day 3 through day 15) significantly attenuated the upregulation of spinal BDNF in the rats with bone cancer-induced pain (t = 3.49, P < 0.01), while no change was observed in the control rats (t = 0.15, P > 0.05; n = 8–9 rats per group); (C) Intrathecal injection of PDTC significantly attenuated the increase of input (stimuli intensity) – output (EPSC amplitude) response in the dorsal horn neurons in the modeled rats (F(1, 19) = 9.76, P < 0.01; n = 9–12 neurons per group); (D) Intrathecal injection of PDTC significantly recovered the ipsilateral paw withdrawal threshold response to mechanical (F(1, 17) = 11.63, P < 0.01) and radiant thermal stimuli (F(1, 17) = 9.81, P < 0.01) in the rats with bone cancer (n = 9–10 rats per group). Control vs. TCI: *, P < 0.05; **, P < 0.01; TCI vs. TCI + PDTC: ?, P < 0.05; ??, P < 0.01.