1：Experimental Design and Method Selection:

The study used in vitro experiments on A431 human squamous cell carcinoma cells to compare HBO combined with ALA-PDT versus control, HBO alone, and ALA-PDT alone. Techniques included CCK-8 assay for cell viability, flow cytometry for apoptosis, western blotting for protein expression, MDC staining and immunofluorescence for autophagy, and ROS detection.

2：Sample Selection and Data Sources:

A431 cell line purchased from ATCC, cultured in DMEM with fetal bovine serum and antibiotics. Cells were seeded in plates and treated with HBO (0.25 MPa; 98% O2 + 2% CO2) for 1 h daily before ALA treatments. ALA was added at concentrations (0,

3：25 MPa; 98% O2 + 2% CO2) for 1 h daily before ALA treatments. ALA was added at concentrations (0, 1, 3, 1, 3 μM) for 48 h, with light exposure using a red light lamp. List of Experimental Equipment and Materials:

0.1, 0.3, 1, 3 μM) for 48 h, with light exposure using a red light lamp. 3. List of Experimental Equipment and Materials: Cell culture equipment, HBO chamber, Phototherapeutics Paterson BL1000A lamp, microplate reader, flow cytometer, fluorescence spectrophotometer, fluorescence microscope, various kits and reagents (e.g., CCK-8 kit, antibodies from Abcam, MDC from Sigma).

4：Experimental Procedures and Operational Workflow:

Cells were treated with HBO and/or ALA, followed by light exposure. After treatments, assays were performed: CCK-8 for viability, flow cytometry with Annexin V/PI for apoptosis, western blotting for protein levels, MDC staining and immunofluorescence for autophagy, ROS detection kit for reactive oxygen species.

5：Data Analysis Methods:

Statistical analysis using Student's t-test for two groups and one-way ANOVA with Dunnett's test for multiple groups, with significance at P<0.05 or P<0.01. Combination index (CI) calculated using CompuSyn software to assess synergism.