Pa1 014 a

For vesicle transfer, 3 x 10⁵ BHY or FaDu cells were seeded in 6-well plates. After 24 h, the wells were washed with PBS and 2 ml fresh medium containing 10% EdFBS was added, supplemented by the EVs isolated from 6 ml conditioned medium from 0 or 6 Gy-irradiated cells. 24 h later, cells were detached with a cell scraper and analyzed via Western Blot.
To confirm EV-uptake into recipient cells, the isolated EVs were stained with green fluorescent dye PKH67 (MINI67, Sigma-Aldrich) as previously described (19 (link)). 40,000 BHY cells were seeded in 12-well silicone grids (Ibidi) on glass slides, after 24 h the wells were washed with PBS and 250 µl new EdFBS-supplemented medium was added, containing PKH67-stained EVs isolated from 750 µl conditioned medium of BHY cells. After 24 h, cells were fixed in 4% paraformaldehyde and nuclei were stained with Hoechst 33342.
For visualization of surface GRP78 after irradiation, cells were seeded on glass slides and after 24 h irradiated with 0 and 6 Gy. Further 24 h later, cells were fixed with 4% paraformaldehyde and labelled with an anti-GRP78 antibody dilution of 1:100 (PA1-014A, Invitrogen) followed by a secondary Alexa Fluor 488-coupled antibody (anti-rabbit: A-11008, Thermo Fisher). Cells were not permeabilized for appropriate labelling of surface GRP78.

Western blot analysis was performed to assess the protein levels of HSPA5, NRF2 and GPx4 in borax‐treated cells. Initially, protein samples (20 μg) were prepared and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The separated proteins were then transferred from the gel to a polyvinylidene fluoride (PVDF) membrane. To prevent non‐specific binding, the PVDF membranes were blocked using 2% bovine serum albumin (BSA). Subsequently, the membranes were incubated overnight at 4°C with primary antibodies specific to HSPA5 (dilution 1 μg/mL; PA1‐014A; Invitrogen), NRF2 (dilution 1:2000; PA5‐88084; Invitrogen), GPx4 (dilution 1:1000; PA5‐102521; Invitrogen) and cleaved caspase‐3 (dilution 1:1000; Cat No. 9661; Cell Signaling). Following the primary antibody incubation, the membranes were washed to remove any unbound antibodies. Next, the membranes were incubated with secondary antibodies. To visualize the protein bands, a chemiluminescence ECL (enhanced chemiluminescence) kit (34579, Thermo Scientific) was used. The emitted light was captured using an imaging system, and the resulting images were analysed using software such as ImageJ.

Tumors, hearts, livers, spleens, lungs, kidneys and brains from the mock-BBZ CAR and Pep42-BBZ CAR groups were embedded in paraffin, and then sliced into sections with a thickness of 4 μm. Sections were deparaffinized, rehydrated, and processed for antigen retrieval. H&E staining was performed on heart, liver, spleen, lung, kidney, and brain tissue sections to observe their structure. The tumor, liver and lung tissue sections were blocked with 10% goat serum and incubated with the indicated primary antibodies at 4 ℃ overnight. Cy3-conjuncted goat anti-rabbit secondary antibody (A10520, Invitrogen), FITC-conjuncted goat anti-rabbit secondary antibody (F-2765, Invitrogen), and Cy3-conjuncted goat anti-mouse secondary antibody were used (A10521, Invitrogen). The primary antibodies used were as follows: anti-GRP78 antibody (PA1-014 A, Invitrogen), anti-CD3ζ antibody (ET1607-20, Huabio), anti-NESTIN antibody (sc23927, Santa Cruz), anti-CD4 antibody (ET1606-31, HUABIO), and anti-CD8 antibody (ET1609-52, HUABIO).