Figure 5.

MMP1 formed a stable structure with PD-L1 in a protein-bound manner, and MMP1 reversed the SPHK1 phenotype. A-C. Co-IP showing the region of PD-L1 bound to MMP1. A. The total protein of SAS/SCC15 cells was immunoprecipitated with anti-MMP1 antibody, followed by immunoblotting with anti-PD-L1 antibody. B. The total protein of SAS/SCC15 cells was immunoprecipitated with anti-PD-L1 antibody, followed by immunoblotting with anti-MMP1 antibody. C. MMP1-related PD-L1 protein stability western blot assay. SAS and SCC15 cells from the protein translation blocker cycloheximide (CHX) treated (sh NC) control and MMP1 knockdown groups were used, and the rate of PD-L1 protein degradation was detected by western blot timing. D. Docking mode and interactions between MMP1 and PD-L1. E-F. Knockdown of MMP1 restored the effect of SPHK1 on apoptosis in Jurkat cells co-cultured with tumor cells. Apoptosis was detected by flow cytometry after activated Jurkat cells were co-cultured with MMP1-specific siRNA or siRNA NC-treated HNSCC cell lines SAS and SCC15 overexpressing SPHK1 for 24 h. The ratio of cancer cells to Jurkat cells was 1:5. Representative images are shown on the left, and quantitative data are shown on the right. Apoptosis of Jurkat cells cultured alone served as a control. G-H. Knockdown of MMPI rescued the ability of SPHK1 to inhibit HNSCC killing by T cells in vitro. The HNSCC cell lines SAS and SCC15 with SPHK1 overexpression were co-cultured with activated human T cells for 48 h after treatment with MMP1-specific siRNA or siRNA NC for 48 h, and the survival rate of cancer cells was determined. Staining of surviving cancer cells with crystal violet. The cancer cell to T cell ratio was 1:3. Representative images are shown on the left, and quantitative data are shown on the right. I. Knocking down MMP1 rescued the reduced IL2 secretion of T cells caused by SPHK1 overexpression. J-K. Knockdown of MMPI rescued SPHK1-induced high PD-L1 levels. After MMP1-specific siRNA or siRNA NC treatment of SPHK1-overexpressing HNSCC cell lines SAS and SCC15 for 48 h, PD-L protein levels were detected using western blotting. L-M. Multiplex immunofluorescence showed that MMP1 knockdown rescued the SPHK1-induced increase in the proportion of PD-L1⁺ cells. Error bars represent the mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. NS: No significance, PD-L1: Programmed cell death ligand 1.