Extended Data Fig. 6: A T0901317/sorafenib therapy results in lipid peroxidation, an ER stress response and macrophage activation in HCCs.
From: LXRα activation and Raf inhibition trigger lethal lipotoxicity in liver cancer
a, Quantification of lipid peroxidation in MycOE;NrasG12V;Cdkn2aARF–/– cells, upon 3 days of treatment with sorafenib, T0901317 or a combination thereof (by analysing the ratio of green and red fluorescence, values represent mean, ± SD, n = 3 cell cultures per condition, statistical significance was calculated using two-tailed Student´s t test, the analysis was repeated twice with similar results). b, Representative western blot analysis of uncleaved or cleaved Atf6, BiP/HSPA5, P-IRE1α (S724) and Xbp-1s in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, sorafenib or a combination thereof (cropped blot images, n = 3 independent experiments). α-tubulin was used as a loading control. c, Representative western blot analysis of eIF2α, P-eIF2α (S51), GADD34, BiP/HSPA5 and P-IRE1α (S724) in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, BI-882370 or a combination thereof (cropped blot images, n = 3 independent experiments). α-tubulin and Vinculin were used as loading controls. d, Representative western blot analysis of MLKL, P-MLKL (S345), RIPK3, P-RIPK3 (S232) and AIF (nuclear and cytoplasmic fraction) in MycOE;NrasG12V;Cdkn2aARF–/– cells after 3 days of treatment with T0901317, sorafenib or a combination thereof (cropped blot images, n = 3 independent experiments). Vinculin and Histone H3 were used as loading controls. e, FACS based analysis of myeloid cells in MycOE;NrasG12V HCCs upon treatment with T0901317/sorafenib or the corresponding monotherapies (3 d). f-h, Flow cytometry gating strategy. The following populations are identified: immature myeloid cells (iMC: CD11b+,Ly6G+/High,F4/80−, fraction 1), monocyte-macrophages (Mac-mono: CD11b+,Ly6G+,F4/80High, fraction 2) and Kupffer cells (Ku: CD11b+,Ly6G+,F4/80High, fraction 3). Monocyte and macrophages are further distinguished/labeled as Ly6C+ (fraction 2a) or Ly6C− (fraction 2b). Samples were gated on viable leukocytes by DAPI exclusion and doublets were excluded using height versus area dot plots. (shown are representative pseudocolor plots, n = 3 mice per group). i,j, Results of FACS analysis in MycOE;NrasG12V HCCs upon 3 days of treatment with T0901317/sorafenib, T0901317, sorafenib or carrier. Charts show the percentage mean ± SD of monocyte-macrophages (Mac−mono: CD11b+,Ly6G+,F/480+, i), Kupffer cells (Ku: CD11b+,Ly6G+,F4/80High, i) and immature myeloid cells (iMC: CD11b+, j) (n = 3 mice per group, statistical significance was calculated using two-tailed Student´s t test, the experiment was repeated with similar results). k, Treatment of MycOE;NrasG12V;Cdkn2aARF–/– cells for 4 days with T0901317 and/or the Bcl-2 inhibitor ABT-199 (quantification of viable cells by crystal violet staining, values represent mean ± SD, n = 3 independent experiments, statistical significance was calculated using two-tailed Student´s t test). Unprocessed images of the blots and numerical source data are provided as source data files.
