Journal: bioRxiv
Article Title: EGFR/TBK1-dependent mitochondrial quality control contributes to acquired resistance to temozolomide
doi: 10.64898/2025.12.19.695123
Figure Lengend Snippet: A. U251 cells were treated by TMZ. Cells were harvested, fixed and stained by pTBK1 antibody coupled to fluorescent secondary antibody. pTBK1 specific fluorescence was detected by flow cytometry and expressed as a ratio to D0 fluorescence (*: p<0.05 vs D0). B. EGFR phosphorylation was measured by flow cytometry by the use of phospho-specific antibodies (pEGFR Y845 or pEGFR Y1068) on U251 or U251 Rho0 cells. Fluorescence was normalized to the fluorescence measured at D0 (*: p<0.05, **: p<0.01). C. EGFR Y845 as measured as in B. in the presence or absence of NAC 5mM (*: p<0.05, **: p<0.01). D. Phosphorylation of EGFR (Y845 and Y1068) and TBK1 was measured as in B in the presence or absence of PP1 (Src inhibitor). E. Src and TBK1 phosphorylation was measured as in B in U251 and U251 EGFR-cells treated by TMZ for the time indicated on the graph (*: p<0.05, **: p<0.01, ***: p<0.001). F. U251 and U251 EGFR-cells were treated by TMZ 50µM twice a week and cells were counted by flow cytometry at each time point. Cell number was normalized by the cell count at D0. G. The sensitivity of U251 cells to erlotinib was evaluated by MTT during TMZ treatment and the IC50 was calculated by AAT Bioquest “IC50 Calculator” tool ( https://www.aatbio.com/tools/ic50-calculator ).
Article Snippet: TOM20 #612278, BD (Le Pont de Claix, France); TFAM #8076, Cell Signaling Technology (Danvers, MA, USA); EGFR #4267, Cell Signaling Technology; pEGFR Y845 #6963, Cell Signaling Technology; pEGFR Y1068 #3777, Cell Signaling Technology; TBK1 #3504, Cell Signaling Technology; pTBK1 #5483, Cell Signaling Technology; pSrc #sc-81521, Santa Cruz Biotechnology (Dallas, TX, France); VDAC #ab14734 Abcam, (Cambridge, UK); IP3R #sc-28614, Santa Cruz Biotechnology; LC3 #83506, Cell Signaling Technology or #L7543, Sigma Aldrich.
Techniques: Staining, Fluorescence, Flow Cytometry, Phospho-proteomics, Cell Counting
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