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‐κB activation is downstream effector of CXCR5/PLCβ/PKCα/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‐κB activation was monitored by phosphorylation of p65. The p65 and β‐Actin were used as internal control B. C, The CL1‐0 lung cancer cells were transfected with NF‐κB 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‐κB activation. D, The CL1‐0 lung cancer cells were transfected with NF‐κB 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‐κB activation. E, The CL1‐0 lung cancer cells were cotransfected with NF‐κB luciferase reporter vector in the presence of CXCR5, PLCβ, PKCα 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‐κB 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