Journal: bioRxiv

Article Title: A novel, RAS-independent role for NF1 in microtubular dynamics and damage repair dictates sensitivity to T-DM1 in HER2-positive breast cancer

doi: 10.1101/2023.12.06.569572

Figure Lengend Snippet: a-c CETSA for DM1 in BT-474 WT and BT-474 KO cells. a experimental design. Cells are harvested and incubated in suspension with DM1 for 2h and then exposed to increasing temperatures as indicated b western blot for β-tubulin of cell extracts from BT-474 WT and BT-474 KO cells . c. Quantification of band intensities from CETSA western blots (n = 3 independent experiments), normalised on baseline signal at 57°C. Ribbons indicate SD. We used 2-way ANOVA separately on WT and KO to test for significance of temperature and drug. We observed that temperature was significant both in the WT and in the KO case (pvalue=2.05x10 - and 1.79 x10 -9 , respectively). However, the addition of DM1 was significant only in the KO case (pvalue=4.62 x10 -3 , 0.78 in the WT case). d. Representative kymographs of microtubules grown in vitro with or without NF1 at indicated concentration and 1μM DM1 from GMPCPP-stabilized seeds and followed by TIRF microscopy e. NF1 increases the fraction of microtubules initiating elongation in the presence of 1μM DM1. The percentage for each condition was calculated as the number of GMPCPP seeds elongating divided by the total number of seeds present in the acquired ROI. f. Quantification of growth rate of microtubules grown with 1μM DM1 and with 100nM or 300nM NF1. g. Quantification of depolymerization rate of microtubules grown with or without 100nM NF1 and with or without 1μM DM1. Data are means ± SEM. Kruskal-wallis test with Dunn post-hoc test was used to assess significance. h. Single frames of time-lapse videos of taxol-stabilized rhodamine-labeled microtubules incubated with 1μM DM1 i. Taxol-stabilized rhodamine-labeled microtubules were first incubated without Taxol and tubulin for 1.5 min and subsequently with 5 mM HiLyte Fluor-488-labeled tubulin with 100nM NF1. After 10 min, the residual free green tubulin was washed out with the wash buffer supplemented with 25% glycerol to prevent microtubule depolymerization, in order to better visualize incorporation of green tubulin. Kymograph showing green tubulin incorporation sites into Rhodamine-labelled microtubule lattices (magenta). Arrows indicate complete repair. j. reconstruction of microtubule tip trajectories after monitoring of microtubule elongation in BT-474 WT and BT-474 KO cells transiently transfected with an EB3-GFP-expressing vector and monitored by live cell imaging for 1 minute with acquisitions every 4 seconds. Microtubule tip fates were reconstructed by the TrackMate ImageJ plugin. k quantification of tip mean speed. At least 1000 events from at least 3 cells were calculated per condition. 2-way ANOVA with Tukey’s HSD. **** p < 0.001. l-m immunofluorescence for acK40 alpha tubulin in HCC1954 WT and HCC1954 KO . m quantification of mean intensity normalized by area occupied by cell cytoplasm. 4 fields of view per condition

Article Snippet: Cells were blocked with 5% BSA in PBS for 30 minutes and then incubated with primary antibody anti-HER2 Alexa Fluor-488 conjugate (#FAB1129G-025, Biotechne) diluted in 1% BSA in PBS for 1 hour at 4° C in agitation.

Techniques: Incubation, Suspension, Western Blot, In Vitro, Concentration Assay, Microscopy, Labeling, Transfection, Expressing, Plasmid Preparation, Live Cell Imaging, Immunofluorescence