Fig. 4
The expression of SPP1 in HNSCC positively correlates with P4HA1. (A) The box plot illustrates the distribution of P4HA1 gene expression in normal tissues and HNSCC tissues from the TCGA-HNSCC cohort, and the differences were analyzed using the Wilcoxon rank-sum test (P < 0.001). (B) The box plot displays the abundance of SPP1 + macrophage infiltration in the high P4HA1 expression group and low P4HA1 expression group (cut-off = 0.5) among 502 TCGA-HNSCC patients, and the differences were analyzed using the Wilcoxon rank-sum test (P < 0.001). (C) Kaplan-Meier survival analysis was conducted on HNSCC samples (n = 499), and the log-rank test revealed a significant difference (P < 0.0001). (D) The macrophages were treated with SCC25 or CAL27 conditioned media for 48 h. And then the expression of SPP1 in the macrophages was detected using cell immunofluorescence (left panel), and the cell skeleton was labeled using F-actin (left panel). The proportion of macrophages with SPP1 expression was quantified relative to the total number of macrophages (right panel). (E) Immunofluorescence staining was performed to detect the levels of SPP1 in macrophages after 72 h of indirect co-culture with SCC25 (transfected with si-Ctrl or si-P4HA1), and the proportion of macrophages with SPP1 expression was quantified relative to the total number of macrophages. (F) Transwell migration and invasion assays were conducted to assess the number of tumor cells at the bottom of the chamber within 24 h after addition of conditioned medium of macrophages. (G) Wound healing assay was performed to detect the migration of tumor cells. Error bars, SEM, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.
