Fig. 3: Inhibition on ferroptosis delays cannabichromene (CBC)-induced cell death.

A Annexin V-FITC/propidium iodide (PI) staining analysis confirmed that a ferrostatin-1 and erastin 1 h pre-treatment affected CBC-induced cell death. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. no treatment. B Western blot analysis showing the change in HMOX1 protein expression induced by ferrostatin-1, erastin, and CBC treatment. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. no treatment. C Two siRNAs for HMOX1 were prepared and transfected into both MIA PaCa-2 and PANC-1 pancreatic cancer cells. The western blot image shows that HMOX1 expression was successfully downregulated after siRNA transfection in the two pancreatic cancer cell lines. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s multiple comparison test, with P < 0.01 or P < 0.001 vs. siNT. D Annexin V-FITC/PI staining analysis showed that HMOX1 inhibition affected CBC-induced cell death. Statistical significance was determined using Student’s t tests, with p-values ≤ 0.05 indicating statistical significance. Results are expressed as mean ± SD (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s and Tukey’s multiple comparison test, with P < 0.01 or P < 0.001 vs. siNT. MIA PaCa-2 and PANC-1 cells were treated 35 μM and 30 μM CBC, respectively. Additional treatments included 10 μM Fer-1 and erastin.