Journal: Nature medicine

Article Title: Aurora kinase A drives the evolution of resistance to third generation EGFR inhibitors in lung cancer

doi: 10.1038/s41591-018-0264-7

Figure Lengend Snippet: a Immunoblot analysis of total and phosphorylated AURKA in PC9 and H1975 parental and acquired resistant cell lines treated with 1uM of the indicated inhibitors for 24 h. b Mean of cell proliferation of PC9 or H1975 cells transfected with plasmids expressing the indicated genes and treated 1uM EGFR-TKI for 72 h compared to DMSO treated cells performed in n=3 biologically independent samples. Significance based on comparison to LacZ control. c Immunoblot analysis 4 parental and 8 acquired resistant cell lines. Quantified intensities for pAURKA and TPX2 relative to the parental cell line is shown. d Proliferation compared to DMSO of PC9 and H1975 parental or acquired resistant cells treated with 1uM of osimertinib or rociletinib, 30nM of MLN8237 or the combination for 72 h. Mean over n=3 biologically independent samples. e Apoptosis measured by YO-PRO-1 positivity in the same models and drug treatments for 72 h. Shown is mean from n=3 biologically independent samples. f Mean tumor volume (mm 3 ) of PC9-RR xenografts during treatment with rociletinib (100mg/kg), MLN8237 (10mg/kg) or the combination. n=10 tumors in the vehicle and rociletinib arm, and n=7 in the MLN8237 and combination arm. g Percent change in tumor volume compared to baseline for individual PC9-OR cell xenografts teated for 11 days with osimertinib (5mg/kg), MLN8237 (10mg/kg) or the combination. P-value comparing combo to single agent osimertinib treatment. h Immunoblot of lysates from parental PC9, PC9-OR and PC9-RR cells treated with the indicated inhibitors or DMSO for 24 h. i Proposed mechanism for the efficacy of the combination in acquired resistant cells. P-values based two-tailed Student’s t-test. Error bars represent s.e.m. Blots are representative of at least two independent experiments. Full blots shown in .

Article Snippet: Antibodies for pEGFR (Y1068; 3777), pERK1/2 (T202/Y204; 4370), ERK1/2 (9102), pAKT (S473; 4060), AKT (9272), PARP (5625), pAURKA (T288; 3079), AURKA (4718) pRb (S780; 9307), BIM (2933), pBIM (S69; 4585), BAX (5023), Vimentin (5741), p-p65 (S536; 3033), p65 (8242), Pan phospho-AURKA/B/C (T288/T232/T198; 2914), CD44 (3570), Histone H3 (9715) were purchased from Cell Signaling Technology; TPX2 (HPA005487) and pan total AURKA/B/C (HPA002636) were purchased from Sigma; EGFR (SC-03), NEDD9 (sc-33657), AJUBA (sc-398008), PAK1 (sc-16617), CD24 (sc-19585), CD133 (sc-30219), B-tubulin (sc-9104), p53 (sc-126) were purchased from Santa Cruz biotechnology and FZR1/CDH1 (ab3242) from Abcam and V5 tag (46–0705) from Thermofisher.

Techniques: Western Blot, Transfection, Expressing, Comparison, Control, Two Tailed Test

Journal: Traffic (Copenhagen, Denmark)

Article Title: Spastin Couples Microtubule Severing to Membrane Traffic in Completion of Cytokinesis and Secretion

doi: 10.1111/j.1600-0854.2008.00847.x

Figure Lengend Snippet: A–F) In HeLa cells fixed with methanol, endogenous spastin (A and D) colocalizes with the midbody markers Aurora B (B) and PRC1 (E). Note the double-ring appearance of spastin on either side of the stembody, most obvious in (A). G–I) Spastin (G) also colocalizes with GFP-VPS4A(E235Q) (H) in double-ring structures in the midbody, in methanol-fixed HeLa cells.

Article Snippet: Rabbit polyclonal anti-GFP (6556), rabbit polyclonal anti-aurora B and rat polyclonal anti-tyrosinated tubulin (YL1/2) were obtained from Abcam.

Techniques:

Journal: Traffic (Copenhagen, Denmark)

Article Title: Spastin Couples Microtubule Severing to Membrane Traffic in Completion of Cytokinesis and Secretion

doi: 10.1111/j.1600-0854.2008.00847.x

Figure Lengend Snippet: A–C) Hela (A and B) and MRC5 (C) cells were labeled with alpha-tubulin following spastin depletion with pooled siRNA oligonucleotides 1–4. Note long intercellular bridges (arrowheads) that were sometimes very convoluted (B). Alpha-tubulin-labeled puncta were often seen in association with these bridges (arrow in B). D and E) The intercellular bridges (arrowheads) were also seen following spastin depletion using two individual spastin siRNA oligonucleotides. Successful spastin depletion in these experiments is verified in (F). G–I) The intercellular bridges (arrowheads) typically joined two cells, as shown in DIC image (G) of YFP–tubulin (H)-expressing HeLa cells depleted of spastin. J–L) Some of the intercellular bridges had the appearance of very elongated midbodies, which labeled with midbody markers [e.g. aurora B; (K)] as well as with MT markers (J). M–O) Hela cells transfected with 60 kD myc-spastinK388R (M) and labeled for alpha-tubulin (N) also displayed similar intercellular bridges (arrowhead). Formaldehyde was used in fixed preparations except (J–L) where methanol was used.

Article Snippet: Rabbit polyclonal anti-GFP (6556), rabbit polyclonal anti-aurora B and rat polyclonal anti-tyrosinated tubulin (YL1/2) were obtained from Abcam.

Techniques: Labeling, Expressing, Transfection