Ab91654

Antibodies against CRTC3 (ab91654, WB, 1:1000), SLC7A11 (ab216876, WB, 1:1000), GPx4 (ab125066, WB 1:1000, IHC 1:100), and β-actin (ab8227, WB, 1:1000) were purchased from Abcam. Sorafenib (S7397), erastin (S7242), RSL3 (S8155) and Ferrostatin-1 (S7243) were purchased from Selleck Chemicals. PRGL493 (HY-139180) was purchased from MedChemExpress. IFN-γ (300-02) was purchased from PEPROTECH.

Panc02 cells were added to 35 mm confocal dishes (1 × 10⁵/well) and cultured for 12 h at 37 °C. Cells were then treated for 8 h with either PBS or TiSe₂ nanosheets (25 µg/mL), after which they either were or were not subjected to US irradiation (1.0 MHz, 0.5 W/cm², 50% duty cycle, 1 min). Following an additional 24 h incubation, cells were fixed for 15 min with 4% paraformaldehyde (PFA) in PBS at room temperature, rinsed three times using PBS, and stained overnight with anti-CRT (Abcam, Cambridge, UK, ab91654) at 4 °C. Following three additional washes with PBS, cells with incubated with AF594-conjugated secondary antibodies and Dio-488 (to stain the cell surface) for 1 h at room temperature. Nuclei were then stained using DAPI, and CRT exposure was visualized by CLSM.
Cells were additionally harvested for flow cytometric analyses of CRT surface exposure. Briefly, treated cells were collected, rinsed three times, and incubated for 1 h on ice with primary anti-CRT in 2% FBS. Cells were then rinsed with PBS and probed with an AF594-conjugated secondary antibody in 2% FBS for 1 h. Samples were then analyzed via flow cytometry to detect cell surface CRT exposure.