*4.7. Western Blot Analysis*

Control or treated groups were dry pelleted after 48 h of treatment and lysed with equal volumes of 1× lysis buffer (1 mM PMSF and 1 X protease Inhibitor Cocktail) on ice for 30 min. Then, they were sonicated and centrifuged at 18000 G for 20 min at 4 ◦C. Protein concentrations were determined by Bradford protein assay. Equal amounts of proteins (30 μg) were separated by 12% SDS PAGE gel electrophoresis and then transferred to PVDF membranes that were blocked in PBS 5% BSA containing 0.1% Tween 20 at room temperature for 1 h. Afterward, the membranes were incubated with primary antibodies against PD-L1 (1:200) (Santa Cruz: Biotechnology) or α-tubulin (1:5000) (Cell Signaling) overnight at 4 ◦C. The next day, membranes were washed (PBS-0.1% Tween) and incubated with HRP-secondary antibody (1:5000) at room temperature for 1 h. After washing, the protein bands were detected with a chemiluminescence detection system (ChemiDocTM Touch Gel Imaging System—Bio-Rad Laboratories), which were quantified and numerated using Fiji software (Rasband, W.S., ImageJ). A ratio was calculated for PD-L1 expression/αtubulin expression, and then a second ratio was calculated for test/control to compare expression of treated to untreated cells.

### *4.8. PD-L1 Expression Analysis: Immunofluorescent Staining and Flow Cytometry*

For surface labeling, the same protocol of seeding and treatment as described above for cell cycle analysis was followed. LCLs and DLBCLs cells were collected and washed with DPBS. Then, they were labeled for 15 min in the dark at RT with anti-PD-L1-PE (Biolegend) (Table S1). Intracellular PD-L1 staining was performed on LCLs and DLBCLs cells treated with native fucoidan or F1/F2 fractions using the IntraPrep Permeabilization Reagent kit (Beckman Coulter) according to the protocol recommended by the supplier. Acquisitions were performed on FACSCalibur. Results were analyzed with Kaluza Analysis 2.1 Software. Fold change was calculated based on the mean fluorescence intensity ratio of PD-L1 on its isotypic control, and then normalized to the control (untreated cells).
