Fig 2: NAMPT was a downstream target of miR-127-5p. a The predicted binding sites and its mutant for miR-127-5p on the 3′UTR of NAMPT were shown. b Relative luciferase activity was determined in OA chondrocytes co-transfected with NAMPT-wt or NAMPT-mut and NC or miR-127-5p. c The enrichment of NAMPT was measured by RIP assay in OA chondrocytes transfected with NC or miR-127-5p. d, e The level of miR-127-5p and protein level of NAMPT were determined by qRT-PCR and western blot analyses, respectively, in OA chondrocytes transfected with NC, miR-127-5p, anti-NC, or anti-miR-127-5p. f The protein expression of NAMPT was examined by western blot assay in OA chondrocytes transfected with si-NC, si-circ_0128846#1, si-circ_0128846#1 + anti-NC, or si-circ_0128846#1 + anti-miR-127-5p. g, h NAMPT mRNA and protein levels were measured in OA cartilage tissues and normal cartilage tissues by qRT-PCR and western blot analyses, respectively. i The association between NAMPT mRNA level and miR-127-5p abundance was analyzed in OA cartilage tissues. j The correlation between circ_0128846 level and NAMPT mRNA level was analyzed in OA cartilage tissues. *P < 0.05

Fig 3: Modulation of the Wnt/β-catenin signaling pathway caused by NAMPT inhibition are NMN-dependent. a TOPFlash assay of LoVo or HCT116 cells treated with 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. b The expression of β-catenin and cyclin D1 in LoVo cells with shCtrl + 2% DMSO, shNAMPT + 2% DMSO, or shNAMPT + NMN (100 μM) was analyzed by western blotting. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. c Western blot analysis of whole cell lysates from LoVo cells exposed to 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days showed the levels of β-catenin and cyclin D1. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. d Western blot analysis of whole cell lysates from HCT116 cells exposed to 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days showed the levels of β-catenin and cyclin D1. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. e The cytoplasmic and nuclear expressions of β-catenin in LoVo cells treated with 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days were analyzed by western blotting. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. f The cytoplasmic and nuclear expression of β-catenin in LoVo cells with shCtrl + 2% DMSO, shNAMPT + 2% DMSO, or shNAMPT + NMN (100 μM) was analyzed by western blotting. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. g The expression of β-catenin and cyclin D1 in LoVo cells with shCtrl + 2% DMSO, shNAMPT + 2% DMSO, shCtrl + CHIR99021 (100 ng/mL), or shNAMPT + CHIR99021 (100 ng/mL) was analyzed by western blotting. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the control group

Fig 4: Knockdown of Axin can counteract the growth inhibition induced by NAMPT depletion via Wnt/β-catenin signaling activation. a Western blot analysis of whole cell lysates showed the protein levels of Axin from LoVo cells exposed to 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. b Western blot analysis of whole cell lysates showed the protein levels of Axin from HCT116 cells exposed to 2% DMSO, FK866 (10 nM), or NMN (100 μM) + FK866 (10 nM) for 2 days. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. c The expression of Axin in LoVo cells with shNAMPT or shCtrl was analyzed by western blotting. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. d The efficiency of Axin knockdown in LoVo cells was determined by western blot analysis. The data are presented as the mean ± SD of three independent experiments. Student’s t-test was used for statistical analysis. The expressions of β-catenin from whole cell lysates (e) and cytoplasmic lysates (f) of LoVo cells with shAxin + 2% DMSO, shCtrl + 2% DMSO, shAxin + FK866, or shCtrl +FK866 were analyzed by western blotting. Student’s t-test was used for statistical analysis. g The viability of LoVo cells with shCtrl + 2% DMSO, shCtrl + FK866, shAxin + 2% DMSO, or shAxin + FK866 was analyzed using a CCK-8 assay. Student’s t-test was used for statistical analysis. h Overall survival (OS) analysis of CRC patients based on Axin expression was analyzed by Kaplan-Meier plotter. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with the control group

Fig 5: Knockdown of NAMPT suppresses CRC cell growth in vivo. a-d The effect of NAMPT expression on in vivo tumorigenicity was evaluated using a xenograft nude mouse model. a Tumors formed in nude mice; b dissected subcutaneous tumors, scale bars: 10 mm; c curves of tumor growth; d tumor weight; number = five mice/group. The error bars indicate the SD. Student’s t-test was used for statistical analysis. e The protein expression levels of NAMPT in xenograft tumors were analyzed by western blotting. f Quantification of the protein expression levels of NAMPT in xenograft tumors. Student’s t-test was used for statistical analysis. g Representative images of IHC staining for NAMPT in the xenografts. The box graphs show the quantification of IHC staining. Scale bars: 50 μm. Student’s t-test was used for statistical analysis. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the control group