Fig 2: CBD plays protective effects on OM via the Nrf2/Keap1/ARE pathway. HOK cells were transfected with Nrf2-siRNA and Scramble-siRNA, respectively. After 6 hrs transfection, HOK cells were treated with 5-FU and CBD. Then, the cells were collected for the following analysis. (a) Knockdown efficiency of Nrf2 as measured by qRT-PCR. *P < 0.05vs. vehicle group. (b) Detection of Nrf2, HO-1, and NQO1 protein levels by western blotting. ß-Actin was used as the internal control. (c) Grayscale analysis of the blots. (d) The mRNA expression levels of Nrf2, HO-1, and NQO1 as measured by qRT-PCR. ß-Actin was used as the internal control. (e) DCFH-DA staining. Scale bar, 200 µm. (f) Quantitative analysis of the mean fluorescence intensity. Data are expressed as the means ± SD. *P < 0.05vs. the 5-FU alone group; #P < 0.05vs. the 5-FU+CBD group.

Fig 3: Apatinib promotes in vivo ferroptosis of glioma cells. (a) Subcutaneous tumor images of nude mice. (b) The growth curve of subcutaneous tumors. (c) The weights of subcutaneous tumors. (d) Representative hematoxylin-eosin (HE) staining images of subcutaneous tumors. (e) Representative immunohistochemistry images of Ki67 using subcutaneous tumors. Effect of apatinib treatment on subcutaneous tumors, the levels of ROS (f), MDA (g), Fe (h), and GSH (i), expression levels of GPX4 and SLC7A11 (j), and on the expression levels of Keap1, Nrf2, p-VEGFR2, and VEGFR2 (k). *P < 0.05; **P < 0.01.

Fig 4: Nrf2 overexpression reverts the increase in iron, the decrease in GSH, GPX4, and SLC/A11, and the changes in expression of the VEGFR2/Nrf2/Keap1 pathway components induced by apatinib. (a, b) Fe levels; (c, d) GSH levels; (e, f) expression levels of GPX4 and SLC7A11; (g, h) expression levels of Keap1, Nrf2, p-VEGFR2, and VEGFR2 in U251 and U87. *P < 0.05; **P < 0.01.

Fig 5: Apatinib induces ferroptosis of glioma cells. All the data refer to 72 h in the presence and in the absence of apatinib. (a) Flow cytometry representative histograms of DCFH-DA-stained U251 cells. (b) Fold change of the mean fluorescence intensity of DCFH-DA-stained U251 cells. (c) Flow cytometry representative histograms of DCFH-DA-stained U87 cells. (d) Fold change of the mean fluorescence intensity of DCFH-DA-stained U87 cells. (e, f) MDA levels in U251 and U87 cells. (g, h) Fe levels in U251 and U87 cells. (i, j) GSH levels in U251 and U87 cells. (k, l) Western blotting was used to determine the levels of GPX4 and SLC7A11 in U251 and U87 cells. (m, n) Western blotting was employed to detect the expression levels of Keap1, Nrf2, p-VEGFR2, and VEGFR2 in U251 and U87 cells. *P < 0.05; **P < 0.01.