Tween 80

BALB/c female nude mice aged 4-week-old were purchased from SiPeiFu (SPF Biotechnology Co., Ltd, Beijing) and lived in a germfree environment with constant temperature and humidity, suitable for food and water. All mice were slowly injected subcutaneously with U87-MG cells (5 × 10⁶ cells/100 µL) under the armpit of the right forelimb. When the tumors reached 3-4mm in diameter, these nude mice were divided into 5-DMN and control groups, and administered an intraperitoneal (i. p.) injection of 5% DMSO + 40% PEG300 (Selleck) + 5% Tween 80 (Selleck) + 50% water (control) or 5-DMN 3 mg/kg that was dissolved in DMSO, PEG300, Tween 80 and water (5:40:5:50 v/v). Mice were given treatment every other day using 100 μL total volumes. Tumor volumes were assessed every two days and were calculated as 0.5 × length × width². The mice were sacrificed under adequate anesthesia 21 days after treatment.

To investigate the effect of SR1078 on cSCC growth in vivo, we conducted an animal experiment. Firstly, 10 eight-week-old SKH-1 mice were randomly divided into two groups (n = 5 per group). The injected dose of SR1078 was 20 mg/kg, and the weight of each mouse was estimated to an average of 25 g. SR1078 was dissolved in the solution including 10% dimethyl sulfoxide (DMSO, Selleck), 40% PEG300 (Selleck), 5% Tween80 (Selleck) and 45% ddH₂O. The agonist group and control group were injected intraperitoneally with 150 µl SR1078 solution and DMSO solution, respectively. In specific, three times injection in a week was performed before cSCC cell inoculation. Then, XL50 cSCC cells from China Center for Type Culture Collection (Wuhan, China, 5 × 10⁶) and NIH/3T3 cells (mouse embryonic fibroblast, 1 × 10⁶) were injected subcutaneously into the backs of mice to establish a cSCC mouse model. After incubation, mice were injected intraperitoneally with SR1078 or DMSO solution daily. Tumor volume was measured every 2 days and calculated as length × width² × 0.5 (mm³). Eighteen days post-injection, the tumor was harvested for cell proliferation evaluation by IHC of Ki67 expression (MA5-14520, Thermo Fisher Scientific) following the guideline.

The following antibodies from Cell Signaling Technology (CST) were used: Phospho-NF-kappa-B p65 (Ser536) Antibody (3031S), NF-kappa-B p65 (C22B4) Rabbit mAb (4764S), Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (4668), SAPK/JNK Antibody (9252), p38 MAPK (D13E1) XP® Rabbit mAb (8690), Phospho-p38 MAPK (Thr180/Tyr182) (12F8) Rabbit mAb (4631), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (9101S), and p44/42 MAPK (Erk1/2) Antibody (4695). Nrf2 antibody was purchased from Abcam (ab137550), RNA Polymerase II antibody was purchased from Sigma (05-623), and β-action was purchased from Proteintech (60008-1-Ig). RSL3 (S8155) was from Selleck, Ferrostatin-1 (SML0583) was from Sigma, and C11-BODIPY 581/591 lipid peroxidation sensor (D3861) was from Life Technologies. LPS (LPS Ultrapure, Escherichia coli 0111: B4) was from Sigma. PEG300 and Tween 80 were from Selleck.