Fig. 4

SDHB knockout enhanced lipid uptake in GIST cells, which could be inhibited by olverembatinib treatment, and reoverexpression of SDHB increased the resistance of these cells to olverembatinib and abolished the suppression of lipid uptake by olverembatinib. a SDHB knockout enhanced lipid accumulation in GIST-T1 cells. Representative images showing lipid droplets stained with BODIPY-labeled GIST-T1 cells and GIST-T1-SDHB-KO cells. Lipid droplet counts per cell were then measured via ImageJ according to the fluorescence image (n = 6). b Compared with GIST-T1 cells, GIST-T1-SDHB-KO cells were more sensitive to olverembatinib treatment in lipid-depleted medium. GIST-T1 cells and GIST-T1-SDHB-KO cells were treated with 3 nM olverembatinib in RPMI 1640 with 10% or 3% FBS, and then, cell viability was measured with a CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation Assay. c SDH-deficient GIST primary cells cultured in lipid-depleted medium were more sensitive to olverembatinib treatment than those cultured in lipid-rich medium. SDH-deficient GIST primary cells from Patient #1 were treated with 1.25 and 2.5 μM olverembatinib in RPMI 1640 with 10%, 3% and 1% FBS, and then, cell viability was measured with a CellTiter 96^® Aqueous Non-Radioactive Cell Proliferation Assay. d Olverembatinib inhibited lipid droplet accumulation and lipid uptake in SDH-deficient GIST primary cells. Representative images showing lipid droplets stained with BODIPY in primary SDH-deficient GIST (patient #3) cells. Green, BODIPY (493 nm/503 nm) for lipid droplet staining; blue, Hoechst 33342 (350 nm/461 nm) for nuclear staining; red, MitoTracker Red FM (581 nm/644 nm) for mitochondrial staining. e Effects of olverembatinib and other TKIs on lipid uptake in SDH-deficient GIST primary cells from Patient #2. After treatment with DMSO and TKIs at the indicated concentrations for 24 h in basic medium without FBS, the intracellular BODIPY™ FL C16 level in the cells was detected by a multifunction microplate reader at 490 nm/535 nm (n = 4). f Olverembatinib, but not other TKIs, downregulated CD36 in primary SDH-deficient GIST cells. After the cells were treated with the indicated concentrations of olverembatinib, imatinib, regorafenib, and sunitinib for 24 h, the cell lysates were collected for western blot analysis. g Viability response to olverembatinib after SDHB re-expression. Primary SDH-deficient GIST cells from Patients #2 and #3, transfected as in (f), were treated with 100 nM olverembatinib 48 h post-transfection. Cell viability was measured to assess olverembatinib sensitivity before and after SDHB restoration. h Western blot analysis confirmed SDHB overexpression post-transfection. Reoverexpression of SDHB was carried out by transient transfection of pcDNA3.1-SDHB into primary SDH-deficient GIST cells (patients #2 and #3). Western blots showing the overexpression of SDHB after transfection. NC, cells transfected with the pcDNA3.1 vector plasmid. SDHB-OE, cells transfected with the SDHB-overexpressing pcDNA3.1 plasmid. i Lipid uptake response to olverembatinib after SDHB re-expression. Primary SDH-deficient GIST cells from Patients #2 and #3, transfected as in (f), were treated with 100 nM olverembatinib 48 h post-transfection. Lipid uptake capacity was evaluated before and after SDHB restoration. ns, not significant; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001