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

Journal: PLoS ONE

Article Title: Acetate Supplementation Induces Growth Arrest of NG2/PDGFRα-Positive Oligodendroglioma-Derived Tumor-Initiating Cells

doi: 10.1371/journal.pone.0080714

Figure Lengend Snippet: OG33 (A) and OG35 (B-D) cells were induced to differentiate over 6 days in vitro via activation of adenylyl cyclase (1 mM dibutyryl cAMP or 10 μM forskolin) or inhibition of MEK1/2 (1 μM PD035901), ErbB2 (1 μM PD174285), PI3K (5 μM and 10 μM LY294002), or mTOR (10 nM and 20 nM rapamycin [RAP]) signaling. DMSO (0.2% DMSO) was used as a control for PD174285, PD035901 and LY294002 treatments. Despite inhibition of PI3K-Akt-mTOR and ERK signaling pathways, both cells failed to differentiate into cells with oligodendroglial morphology (A, B) or increase immunoreactivity for GPAF or CNPase (not shown). MEK and ErbB2 inhibition actually increased OG35 proliferation in DM. While inhibition of PI3K-Akt-mTOR signaling induced morphological alterations in OG35 cells (B), this was not associated with increased CNPase (C) or ASPA (D) protein levels. In fact, ASPA expression decreased in the presence of 10 μM LY294002 and 20 nM rapamycin. Differentiation of Oli-Neu cells with cAMP for 4 days or inhibition of ErbB2 signaling for 2 days served as controls for oligodendrocyte cell morphology. DM - differentiation medium, SCM - stem cell medium. n = 3 independent cultures. *p < 0.05. Scale bar = 100 μm.

Article Snippet: For pharmacological induction of differentiation, OG33 and OG35 cells were plated in SCM or DM in the absence or presence of dibutyryl cAMP (1 mM in water, Sigma), forskolin (10 μM in ethanol, Sigma), the MEK1/2 inhibitor PD035901 (1 μM in dimethylsulfoxide [DMSO], Tocris Bioscience/R & D Systems; Minneapolis, MN), the ErbB2 inhibitor PD174285 (1 μM in DMSO, Santa Cruz Biotechnology), the PI3K inhibitor LY294002 (at 5 μM and 10 μM in DMSO, Tocris), the mTOR inhibitor rapamycin (at 10 nM and 20 nM in ethanol, Sigma) or 0.2% DMSO as a control (final DMSO concentration of 0.05% for PD174285, 0.1% for PD035901 and LY294002, and 0.2% for PD035901/LY294002/rapamycin treatment).

Techniques: In Vitro, Activation Assay, Inhibition, Control, Protein-Protein interactions, Expressing