Journal: bioRxiv
Article Title: Selective mRNA translation determines adaptative mutability of melanoma cells to anti-BRAF/MEK combination therapy
doi: 10.1101/2025.09.02.673634
Figure Lengend Snippet: A , Schematic representation of the protocol used for the isolation of actively translating ribosomes and their associated transcripts. After a short L-azidohomoalanine (AHA) pulse, newly synthesized AHA-labelled peptides are used to isolate active ribosome complexes through chemical interactions with magnetic beads. Bead-bound complexes are then treated with Proteinase K and the purified RNA is used for RT-qPCR. B , Quantification of 53BP1 mRNA enrichment in active ribosomes performed using RT-qPCR in A375 and M249 control cells (DMSO), in cells surviving targeted therapy (BRAFi/MEKi) and in cells released from the drugs for 9 days (BRAFi/MEKi rel). Data represent the mean ± SEM from 3 (A375) or 4 (M249) independent experiments (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). C , Schematic of the ribosome-bound mRNA mapping (RIBOmap) used to detect translating mRNAs. RIBOmap relies on the use of a tri-probe set: (1) a primer probe that hybridizes to the target mRNAs, (2) a splint DNA probe that hybridizes with the ribosomal 18S RNA, and (3) a padlock probe. When in proximity, the tri-probes produce DNA amplification signals corresponding to active mRNA translation. D , Representative confocal images of translating 53 BP1 mRNAs (magenta) in A375 cells transfected with control siRNA (siCtl) or with siRNA targeting 53BP1 (si53BP1) for 48 hours. Nuclei are stained with DAPI (blue). Scale bar: 10 µm. E , Quantification of translating 53BP1 mRNAs (spots) in the condition described in D. Data shown represent the mean ± SEM of the number of spots/cells quantified in 1 independent experiment (see also for the other biological replicates) (*** p ≤ 0.001). F , Representative confocal images of translating 53 BP1 mRNAs (magenta) assessed by RiboMap assay in A375 control cells (DMSO) or in cells surviving targeted therapy (BRAFi/MEKi). Nuclei are stained with DAPI (blue). Scale bar: 10 µm. G , Quantification of translating 53BP1 mRNAs (spots) in the condition described in F. Data shown represent the mean ± SEM of the number of spots/cells quantified in 1 independent experiment(see also for the other biological replicates) (*** p ≤ 0.001). H , Representative confocal images of translating 53BP1 (magenta) and IQGAP 1 mRNAs (green) assessed by RiboMap assay in A375 cells surviving targeted therapy. Nuclei are stained with DAPI (blue). Scale bar: 10 µm. I , Scatterplot illustrating the correlation between translating 53BP1 mRNAs and IQGAP1 mRNAs in A375 drug tolerant cells. Pearson correlation coefficient (r) and p-value are reported. The data shown are the results of 1 biological replicate (see for the results obtained in other biological replicates). J , Multiplex immunostaining (CODEX) showing the co-expression of 53BP1, CCSER2, HIPK2, IQGAP1, APC, and ANK2 in A375 cells. Scale bar 20 µm. K , Heatmap generated from multiplexed imaging analysis, displaying single-cell mean intensity values (rows) for protein expression of selected differentially expressed markers (columns), regulated at either the translational or transcriptional level in control cells (DMSO) and drug-tolerant cells (BRAFi/MEKi).
Article Snippet: Frozen blocks were sectioned using a cryostat, and tissue sections were mounted onto poly-L-lysine–coated coverslips Akoya Biosciences CODEX multiplex immunostaining was performed according to its manufacturer’s.
Techniques: Isolation, Synthesized, Magnetic Beads, Purification, Quantitative RT-PCR, Control, DNA Amplification, Transfection, Staining, Multiplex Assay, Immunostaining, Expressing, Generated, Imaging
