Supplementary Figure S2 from A PERK-Specific Inhibitor Blocks Metastatic Progression by Limiting Integrated Stress Response–Dependent Survival of Quiescent Cancer Cells

Supplementary Figure S2. (a) Normalized area of single macro-metastases in lung in vehicle (N=22) and HC4-treated animals (N=15). P by MannWhitney test. (b) 2.5x105 HNSCC T-Hep3 cells as single cell suspensions were injected via tail vein in Balb/c nu/nu mice and 24 h after injectionanimals were treated with vehicle or HC4 (50 mg/kg) for 10 days. Frequency of T-HEp3 DCCs in the lung detected via a human specific anti-vimentinantibody was scored. 5 lung sections/animal ± s.d (N=5 per group). Scale bar, 10 µm. P by Mann-Whitney test. (c) Balb/c syngeneic EMT6 micewere treated with HC4 at 10 mg/kg for 17 days. Metastatic lesions were detected by H&E staining and quantified in 5 lung section/animal. P byMann-Whitney test. (d) Quantification of circulating tumor cells/ml blood by HER2 staining of cytospins (N=4 per group) P by Mann-Whitney test. (e)Percentage P-Rb+ micro-metastasis per lung section/animal in vehicle (N=4) and HC4-treated animals (N=6). Scale bar, 25 µm. P by Student’s ttest. (f) Number of D-Hep3 cells per nodule in vehicle and HC4-treated (20 µM, 6 days) CAM tumors (N=6 per group) (left); quantification andrepresentative images of D-HEp3 CAM tumors stained for cleaved caspase-3 (green), vimentin (red), and DAPI (blue) (right). Approximately 1000cells per group were assessed, p by Mann-Whitney test. Scale bars, 25 μm. (g) Mice were injected with MMTV-HER2 cells from primary tumorlesions via the tail vein, allowed them to expand into established metastatic lesions for 3 weeks and treated with vehicle or HC4 (50 mg/kg) for 2weeks. Micro-metastasis were detected by H&E or IF (HER2 detection) staining and quantified in 5 lung section/animal ±s.d (N=8 per group). P byMann-Whitney test