Fig 1.
Adeno-cre induced murine lung cancer model.
A. Experimental design. B. Representative MRI images of murine lungs and tumors (arrows) from the vehicle and STA-8666 treatment cohorts. C. Quantified tumor volume determined by MRI in vehicle versus STA-8666 treated mice. D. Body weight dynamics in mice receiving vehicle or STA-8666.
Fig 2.
STA-8666 treatment prevents tumor growth in vivo.
A. Representative H&E stained lungs. Arrows indicate tumors. Magnification: 4x. Scale bars: 2mm, top panels; 850 μm, bottom panels. B.-D. Quantification of total tumor surface area (B), number of independent tumors (C), and average size of individual tumors (D).
Fig 3.
Signaling consequences of STA-8666 treatment.
A.-F. Left, Representative images and right, quantification of IHC or immunofluorescence for Ki-67 (A), cleaved caspase 3 (B), pS824-KAP1 (C), pT202/Y204-ERK1/2 (D), vimentin (light red) and DAPI (blue) (E) and HSP90 (F). Magnification: 20x. Scale bars: 75 μm, except for vimentin, where scale bar is 40 μm.
Fig 4.
Statistical analysis of correlations in between markers expression in vehicle and STA-8666 treated mice cohorts.
A-D. Shown Spearman nonparametric correlation coefficient with two-tailed p-value between H-score for Ki-67 and tumor area (A), H-score for cleaved caspase 3 and tumor area (B), H-scores for Ki-67 and cleaved caspase 3 (C), and H-scores for Ki-67 and HSP90 (D). Each dot represents an individual mouse. P values shown in red are for STA-8666 treatment cohort, for blue are for vehicle treatment cohort.
Fig 5.
STA-8666 is more effective than irinotecan in controlling the growth of NSCLC cell lines in vitro.
A. Quantification of CellTiterBlue (CTB) viability assay in human A549 and H441 NSCLC cell lines treated with vehicle, STA-8666 and irinotecan in doses of 0.1, 1, 10 μM for up to 5 days. Chart curves represent average data from two independent runs performed with duplicate samples. B-E. Western blot analysis of phosphorylated (p) and total (t) ERK1/2 (B), AKT (C), CHK2 (D) and CDK1 (E) protein expression in human A549 and H441 NSCLC cell lines, at 48 hours after treatment with vehicle (V), STA-8666 and irinotecan in doses of 0.1, 1, or 10 μM, as indicated. Histograms represent data from two independent experiments. All graphs: *, p≤0.05; **, p≤0.01; ***, p≤0.001 relative to vehicle controls.
Fig 6.
Assessment of cell detachment and cell death in STA-8666- and irinotecan-treated NSCLC cell lines in vitro.
A, B. Protein concentration measurements of adherent human A549 (A) and H441 (B) NSCLC cell lines treated with vehicle, STA-8666 and irinotecan in doses of 0.1, 1, 10 μM, at 24, 48 and 72 hrs post drug treatment. Graphs represent average data from two independent runs. C, D. Western blot analysis of PARP cleavage in human A549 (C) and H441 (D) NSCLC cell lines, 48 and 72hrs after treatment with vehicle (V), STA-8666 or irinotecan at doses of 0.1, 1, or 10 μM, as indicated. Histograms represent data from two independent experiments. All graphs: *, p≤0.05; **, p≤0.01; ***, p≤0.001 relative to vehicle controls.
Fig 7.
In vitro benchmarking of STA-8666 and irinotecan for EMT-related markers.
A, B. Western blot analysis of expression of E-cadherin, vimentin, and Snail in human A549 (A) and H441 (B) NSCLC cell lines 48 hours after treatment with vehicle (V), STA-8666 or irinotecan at the doses of 0.1, 1, 10 μM, as indicated. Histograms represent data from two independent experiments, normalized to vehicle (V) controls.