Fig 2: Effects of CXCL14 on the ROS-induced phosphorylation of ERK. (A) H2O2 increased the expression levels of CXCL14 and p-ERK in HCT116 cells treated without or with 15 µM H2O2 or pretreated with 5 mM NAC in a time-dependent manner. (B) Quantitative results of the expression levels of p-ERK. (C) CXCL14 could up-regulated the phosphorylation level of ERK in HCT116/CXCL14 cells. (D) Quantitative results of the p-ERK and total ERK expression levels in the HCT116/CXCL14 cells and HCT/Control cells. (E) CXCL14-deficiency markedly inhibited the phosphorylation of ERK in HCT116/shCXCL14 cells when compared with that in HCT116/shNC. (F) Quantitative results of the p-ERK and total ERK expression levels in HCT116/shCXCL14 and HCT116/shNC cells. All experiments were repeated three times or more. Error bars represent ±S.D. p < 0.05 was considered to be statistically significant. * p < 0.05; ** p < 0.01, *** p < 0.001.

Fig 3: Intracellular sequestration and aggregation of the chemokine receptor Cxcr4 and its ligands, Cxcl14 and Cxcl12, in motoneurons of SLICK-H::Ranbp2flox/flox mice. (A,B) Representative images of Cxcr4, Cxcl14 and Cxcl12 subcellular localizations in YFP+ motoneurons in different genotypes. +/+ mice have weak immunostaining and a diffuse cellular distribution of Cxcr4 and Cxcl14 (A,a-e) and Cxcl12 (B,a-e) in YFP+ motoneurons of the anterior horn, whereas there is intracellular colocalization, sequestration and aggregation of Cxcr4 with Cxcl14 (A,f-j) and Cxcl12 (B,f-j) in YFP+ motoneurons of -/- mice. (f'-j') Magnified images of the outlined regions indicated in f-j. (C) There are no signs of overt glyosis and microglia activation in the anterior horns at d10 (day 10) post-tamoxifen administration, as shown by the absence of GFAP+ and Cd11b+ immunostaining between genotypes. Scale bars: 25 µm. -/-, SLICK-H::Ranbp2flox/flox; +/+, SLICK-H::Ranbp2+/+. Arrows indicate intracellular aggregate or deposit foci.

Fig 4: Differential transcriptome and gene expression analyses of sciatic nerve upon loss of Ranbp2 in motoneurons. (A) Differential RNA-Seq-based whole-transcriptome analysis of sciatic nerves between SLICK-H::Ranbp2flox/flox (-/-), SLICK-H::Ranbp2+/+ (+/+) and TgRBD2/3*-HA::SLICK-H::Ranbp2flox/flox (TgRBD2/3*-HA::-/-) mice. Forty-five upregulated and four downregulated transcripts were found in -/- mice at day 10 post-tamoxifen administration. (B) Validation of ranked RNA-Seq dataset by RT-qPCR and temporal and directional changes of expression of mRNAs between -/- and +/+ mice. Twenty-four transcripts and Frzb were confirmed to be uniquely upregulated and downregulated, respectively, by RT-qPCR in -/- sciatic nerve. Cxcl14 had the strongest upregulation (~10.5-fold) as early as day 3 (d3). *P<0.05, **P<0.01, ***P<0.001, Student's t-test, n=3 or 4 mice/genotype. (C) Temporal and transcriptional changes in expression of Ccl3 in sciatic nerves at d0, d3 and d10. **P<0.01, Student's t-test, n=3 or 4 mice/genotype. (D) Immunoblots (left) and quantification of Cxcl14 (right) from equal amounts of homogenates of sciatic nerves (ScN) and spinal cords (SpC) at d0 and d10. Cxcl14 levels are decreased in ScN and SpC at d10. *P<0.05, Student's t-test, n=4 mice/genotype. Hsc70 is a loading control. All data are expressed as mean±s.d. -/-, SLICK-H::Ranbp2flox/flox; +/+, SLICK-H::Ranbp2+/+; hsc70, heat shock protein 70; d0 and d10 are days 0 and 10 post-tamoxifen administration, respectively.

Fig 5: Effects of CXCL14 on cell proliferation and migration of human colorectal cancer cells. CXCL14 was stably over-expressed in the HCT116 cells, as determined by RT-PCR assay (A) and western blot assay (B). (C) Quantitative results of CXCL14 expression levels. (D) The proliferative capacity of HCT116 cells stably expressing CXCL14 (HCT116/CXCL14) was stronger than that of HCT116/Control only transfected with empty vectors, as measured by colony formation assay. (E) The migratory capacity of HCT116/CXCL14 was stronger than that of HCT116/control cells, as measured by wound healing assay. (F) Quantitative results of cell migration numbers in modified Boyden chamber assays in HCT116/Control cells and HCT116/CXCL14 cells. (G) The expression levels of cell cycle-related and EMT-related proteins in HCT116/CXCL14 cells and HCT/Control cells were measured by western blot. (H) Quantitative results of cell cycle-related protein expression. (I) Quantitative results of EMT-related protein expression. CXCL14 was stably over-expressed in the SW620 cells, as determined by RT-PCR assay (J) and western blot assay (K). (L) Quantitative results of CXCL14 expression levels. M. The expression levels of CDK2, Cyclin A1, vimentin in HSW620/CXCL14 cells, and SW620/Control cells were measured by western blot. (H) Quantitative results of protein expression. The experiments were performed in triplicate (n = 3). Error bars represent mean ± S.D. p < 0.05 was considered to be statistically significant. * p < 0.05; ** p < 0.01, *** p < 0.001.