Journal: Translational Lung Cancer Research
Article Title: TLR7 induces autophagy in non-small cell lung cancer tumor cells and influences anti-tumors responses in patients
doi: 10.21037/tlcr-2025-aw-1173
Figure Lengend Snippet: Study the impact of TLR7 on the autophagy level in lung tumor cells. (A) Example of images of A549 stably transfected with the GFP-LC3 construct (A549 GFP-LC3) treated or not (NT) with an autophagy inducer, the rapamycin (Rapa., 1 µM) or by synthetic TLR7 agonists, CL264 (1 mM) and loxoribin (Loxo., 1 mM) for 6h. The graph represents the quantification of the GFP-LC3 dots per cell in each condition at 6 h post-treatment. The experiment was performed six times. (B) The same experiment as in A was performed with different kinetics: 6, 24, and 48h post-treatments. The graph represents the evolution of the GFP-LC3 dots per cell in each condition over time. The experiment was performed six times. (C) Example of images of A549 GFP-LC3 treated or not (NT) with either inhibitors of the autophagy maturation, Leupeptin and E64d (L + E, 1 ug/mL each), an autophagy inducer (Rapa., 1 µM) or synthetic TLR7 agonists (CL264 and Loxo., 1 mM) for 24 h. The graph represents the quantification of the GFP-LC3 dots per cell in each condition. The experiment was performed three times. (D) Example of images of A549 GFP-LC3 treated or not (NT) with either an inhibitor of the autophagy maturation, the Baf. (100 nM), an autophagy inducer (Rapa., 1 µM) or synthetic TLR7 agonists (Loxo., 1 mM) for 24 h. Lysosomes were marked in red by the addition of lysotracker 90 minutes before the end of the experiment. The graph represents the study of pixel colocalization (green versus red puncta) using the coloc2 script from the ImageJ software. The experiment was performed three times. (E) A549 cells were transfected with a plasmid encoding a dual-labeled LC3 probe, RFP-GFP-LC3. Cells were again treated with Rapa. or with Loxo. (1 mM) for 24 h. Representative images are shown and the number of total autophagic vacuoles (GFP + RFP + + GFP − RFP + ), autophagosomes (GFP + RFP + ), and autolysosomes (GFP − RFP + ) were enumerate. The inset permits visualization of yellow autophagosome (yellow arrow) and red autolysosomes (red arrow). The experiment was performed three times. (F) At the left: measurement of the proportion of lung tumor cells LC3 positive versus negative for both TLR7 low (proportion of lung tumor cells expressing TLR7 <78%) versus TLR7 high (proportion of lung tumor cells expressing TLR7 >78%) NSCLC patients of the cohort 1. In the right: implementation of an autophagy score (score 1, proportion of lung tumor cells LC3 pos <25%; 2, 25%< x <50%; 3, 50%< x <75% and 4, x >75%). Evaluation of the TLR7 expression level in function of the autophagy score. (G) The same analysis as in E was carried out on the NSCLC patients from cohort 2, those treated by neoadjuvant chemotherapies. Student’s t -test, *, P<0.05. Baf., bafilomycin; GFP, green fluorescent protein; LC3, light chain 3; Loxo., loxoribin; NSCLC, non-small cell lung cancer; Rapa., rapamycin; RFP, red fluorescent protein; TLR7, toll-like receptor 7.
Article Snippet: A1 (100 nM, Sigma) or a combination of leupeptin hemisulfate (1 μg/mL, Invitrogen) and E64D (1 μg/mL, Invitrogen).
Techniques: Stable Transfection, Transfection, Construct, Software, Plasmid Preparation, Labeling, Expressing
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