Fig 2: NF-?B activation is responsible for CXCL13-induced cell migration and VCAM-1 expression in lung cancer cells. A-C, The CL1-0 lung cancer cells were pre-treatment with pathway inhibitors of NF-?B (PDTC, 5 µmol/L; TPCK, 5 µmol/L) for 1 h and then performed with cell migration assay A,, qPCR B, and Western blot C, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. D, The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/mL) for different time course (0, 10, 15, 30 and 60 min), the cell lysates were collected, and activation of NF-?B signalling cascade was evaluated by monitoring phosphorylation of IKKa/ß, I?Ba and p65 proteins. The total proteins were used as internal control. E and F, The CL1-0 lung cancer cells were transfected with dominant mutants of IKKa and IKKß for 24 h and then performed with cell migration assay E, and qPCR F, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group

Fig 3: Phospholipase C-ß/PKCa/c-Src signalling pathways are required for CXCL13-promoted cell migration and VCAM-1 in lung cancer cells. A and B, The CL1-0 lung cancer cells were pre-treatment with pathway inhibitors of PLCß, PKCa or c-Src (U73122, 0.5 µmol/L; GF109203X, 3 µmol/L; PP2, 5 µmol/L) for 1 h and then performed with cell migration assay A, and qPCR B, in the presence of CXCL13 (30 ng/mL) for 24 h to evaluate cell migratory ability and VCAM-1 expression, respectively. C, The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/mL) for different time course (0, 10, 15, 30 and 60 min), the cell lysates were collected, and activation of PLCß, PKCa and c-Src signalling pathways was evaluated by monitoring phosphorylation of these proteins. The total proteins were used as internal control. D, The CL1-0 lung cancer cells were transfected with PLCß, PKCa or c-Src siRNAs for 24 h, and the knockdown efficiency was confirmed by using Western blot. E, The CL1-0 lung cancer cells transfected with PLCß, PKCa or c-Src siRNAs were further evaluated cell migration ability E, and VCAM-1 expression F and G, by using Transwell migration assay, qPCR and Western blot, respectively, in the presence of CXCL13 (30 ng/mL) for 24 h. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group

Fig 4: NF-?B activation is downstream effector of CXCR5/PLCß/PKCa/c-Src signal cascade which contributes to VCAM-1 expression and cell migration in lung cancer cells. (A and B) The CL1-0 lung cancer cells were pre-incubated with CXCR5 neutralized antibody (1 µg/mL), U73122 (0.5 µmol/L), GF109230X (3 µmol/L) or PP2 (5 µmol/L) for 1 h, follow by incubated with CXCL13 (30 ng/mL) for 24 h. Then, immunofluorescence staining was performed with anti-p65 antibody. Nuclei were counterstained with DAPI. Representative microscopy images were shown A. The NF-?B activation was monitored by phosphorylation of p65. The p65 and ß-Actin were used as internal control B. C, The CL1-0 lung cancer cells were transfected with NF-?B luciferase reporter vector for 24 h, follow by incubated with CXCL13 (30 ng/mL) for further 24 h. Then, luciferase activity assay was performed to examine NF-?B activation. D, The CL1-0 lung cancer cells were transfected with NF-?B luciferase reporter vector for 24 h, follow by incubated with CXCR5 neutralized antibody (1 µg/mL), U73122 (0.5 µmol/L), GF109230X (3 µmol/L) or PP2 (5 µmol/L) for 1 h, then exposed to CXCL13 (30 ng/mL) for further 24 h. Finally, the luciferase activity was performed to examine NF-?B activation. E, The CL1-0 lung cancer cells were cotransfected with NF-?B luciferase reporter vector in the presence of CXCR5, PLCß, PKCa or c-Src siRNAs for 24 h, follow by incubated with CXCL13 (30 ng/mL) for further 24 h. Then, luciferase activity assay was performed to examine NF-?B activation. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group

Fig 5: Vascular cell adhesion molecule-1 expression is responsible for CXCL13-promoted cell migration in lung cancer. A, The screening of candidate molecules associated with cell migration ability. The CL1-0 lung cancer cells were incubated with CXCL13 (30 ng/mL) for 24 h, follow by collecting RNA sample, then subjecting to qPCR analysis to validate gene expression of candidate molecules which associated with cell migration ability. B and C, The CL1-0 lung cancer cells were incubated with different concentrations (0, 3, 10 and 30 ng/mL) of CXCL13 for 24 h, the RNA or cell lysate were collected, then subjected to analyse gene expression level of VCAM-1 by qPCR and Western blot. D and E, The CL1-0 lung cancer cells were treated with CXCL13 (30 ng/mL) for different time interval (0, 6, 12 and 24 h), the RNA or cell lysate were collected and then subjected to analyse gene expression level of VCAM-1 by qPCR and Western blot. F, The CL1-0 lung cancer cells were transfected with VCAM-1 siRNA for 24 h, and then, the expression level of VCAM-1 was confirmed by Western blot. G, The CL1-0 lung cancer cells transfected with VCAM-1 siRNA were subjected to analyse cell mobility in the presence of CXCL13 (30 ng/mL) by Transwell migration assay. H, The CL1-0 lung cancer cells transfected with CXCL13 shRNA were performed with qPCR to detect VCAM-1 expression level. I and J, The correlation analysis data of CXCL13 and VCAM-1 by using GEPIA bioinformatics tool. The boxplot analysis used log2 (TPM + 1) for log-scale. Results are expressed as the mean ± SD of triplicate samples. *P < .05 compared to the control group. #P < .05 compared to the CXCL13-treated group