Fig 2: Tumour versus normal expression difference of CXCL13 and CXCR5 in LUAD and LUSC. A and B, Expression patterns of CXCL13 and CXCR5 in all lung cancer tissues containing LUAD and LUSC were analysed by using LCE tool and showed by forest plot. SMD: standardized mean difference

Fig 3: 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 4: 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

Fig 5: CXCR5 mediates the VCAM-1 expression and cell migration in response to CXCL13 incubation. A, The CL1-0 lung cancer cells were transfected with CXCR5 siRNA for 24 h, and the knockdown efficiency of CXCR5 was confirmed by Western blot. B and C, The CL1-0 lung cancer cells transfected with CXCR5 siRNA were further performed with cell migration assay B, in the presence of CXCL13 (30 ng/mL) for 24 h, as well as qPCR C, to detect VCAM-1 expression. D and E, The CL1-0 lung cancer cells were pre-incubated with CXCR5 neutralized antibody (0.5 and 1 µg/mL) for 1 h and then performed with cell migration assay D, and qPCR E, 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