Supplemental Material



Supplemental MaterialSupplemental FiguresSupplemental Figure S1. In vivo derivation of lung-tropic sublines from melanoma HTB140 cells. (A) The parental HTB140 cell line (green) and its in vivo selected lung metastatic derivatives were tested for lung metastatic ability. Plots show bioluminescence imaging (BLI) quantification of lung metastatic burden over time (n = 5 mice per cohort). (B) Representative BLI images of the indicated cell lines are shown after tail vein injection. (C) Growth rates as quantified from in vitro proliferation assays of the HTB140 cell line and lung metastatic derivatives LM1a and LM1-744. Average was calculated from seven independent SRB assays. No significance was reached. Data represent the mean ± s.e.m. ****P <0.0001 (Student’s t test).Supplemental Figure S2. Gene Ontology (GO) analyses of breast cancer and melanoma secretomes. (A-B) GO analysis of overrepresented Biological Process (A) and Molecular Function (B) GO Slim terms of all proteins secreted from melanoma cell lines. (C-D) Visualization of GO Biological Process and Molecular Function GO FAT terms after elimination of redundant and child terms obtained from analysis of all proteins secreted by breast cancer (C) and melanoma (D) cell lines. Supplemental Figure S3. Correlation between protein abundance and RNA expression. (A-D) Protein abundance and mRNA abundance ratios were compared for cell lines LM2-A and MDA-MB-231 (A), LM2-B and MDA-MB-231 (B), LM1a and HTB140 (C), and LM1-744 and HTB140 (D). Pearson correlation coefficients (rp) are displayed with corresponding P-values. Corrected Spearman correlation coefficients are 0.35 (A), 0.41 (B), 0.36 (C), and 0.38 (D). Selected proteins are denoted to highlight their differences in protein and mRNA expression levels. Supplemental Figure S4. NID1 interaction map in breast cancer and melanoma secretomes. (A-B) Graphs of interaction networks of NID1 with proteins identified in secretomes of breast cancer (A) and melanoma (B) cell lines. Proteins secreted at least 2-fold higher (red) or lower (green) by sublines compared to their respective parental cell lines are color-coded. Red lines denote direct interactions with NID1; black lines denote interactions among other proteins in the network. Supplemental Figure S5. NID1 overexpression in clonal breast cancer and parental melanoma cell lines promotes lung metastasis. (A-B) Tumor volumes (A) and tumor weights at day 86 post-injection (B) after orthotopic injection of vector and NID1-overexpressing MDA-MB-231 cells (n = 18 - 20). (C) Top: Western blot analysis of NID1 levels in CM of SCP28 vector and NID1-overexpressing cells. Middle: Coomassie blue staining was used as a loading control. Bottom: RT qPCR analysis of NID1 expression relative to parental cell line and normalized to GAPDH levels. (D-E) BLI quantification (D) and representative images (E) of lung metastasis development after t.v. inoculation of vector and NID1-overexpressing SCP28 cells (n = 6). (F) KM representation of OS of mice bearing lung metastases from (D). P = 0.21, n = 9, HR = 2.40 (Cox regression). (G) Western blot analysis of NID1 levels in CM of HTB140 vector and NID1-overexpressing cells. Middle: Coomassie blue staining. Bottom: RT qPCR analysis of NID1 expression relative to parental cell line and normalized to GAPDH levels. (H) BLI quantification of lung metastasis development after t.v. inoculation of vector and NID1-overexpressing HTB140 cells (n = 10). (I) Weights of primary tumors after s.c. injection of control and NID1-overexpressing HTB140 cells (day 68 post-injection, n = 12). Bar-and-whisker plots display the medians, 25th and 75th percent quartiles, and the full range of variation (from minimum to maximum). The P-value was calculated using the Mann-Whitney test. Tumor progression data are displayed as the mean ± s.e.m (n = 12) and P-values were calculated using the Student’s t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Supplemental Figure S6. NID1 knockdown in lung-metastatic LM1-744 melanoma cell line reduces lung metastasis in vivo. (A) Top: Western blot analysis of NID1 levels in CM of control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2) LM1-744 cell lines. Middle: Coomassie blue staining was used as a loading control. Bottom: RT qPCR analysis of NID1 expression relative to parental cell line and normalized to GAPDH levels. (B-C) BLI quantification (B) and representative images (C) of lung metastasis development by vector and NID1 knockdown (NID1-KD2) LM1-744 cell lines (n = 5 - 6). Data represent the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student’s t test). Supplemental Figure S7. NID1 promotes cancer cell migration, invasion, and adhesion to endothelial cells in vitro. (A-B) Transwell migration (A) and invasion (B) assays of HTB140 vector and NID1-overexpressing cells. (C-D) Transwell migration (C) and invasion (D) assays of LM1-744 control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2). (E-H) Attachment to HPMEC-ST1.6R endothelial cell (EC) monolayers by vector or NID1-overexpressing SCP28 (E) and HTB140 (F) cells, and control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2) LM1a (G) and LM1-744 (H) cells. (I) Attachment of HTB140 vector and NID1-overexpressing cells to monolayers formed by microvascular ECs (HPMEC), lung epithelial cells (HPL1), and plastic plates. Attachments are normalized to the number of attached vector control cells in each condition (n.s. = not significant). (J) Attachment of HTB140 vector and NID1-overexpressing to ECs after treatment of cancer cells with serum free media (SFM) or CM from vector or NID1-overexpressing cells. (K-M) Immunofluorescence images (K) and quantification of vascular permeability by area occupied by diffused dextran (L) and the number of leaked sites (M) after treatment of mice with CM derived from vector and NID1-overexpressing HTB140 cells (n = 5 mice). Scale bar 100?m. (N) Transendothelial cell migration through a EC monolayer of vector and NID1-overexpressing MDA-MB-231 cells. In vitro assays were performed at least in triplicate. (O-Q) Immunofluorescence images (O) and quantification of vascular permeability by area occupied by diffused dextran (P) and the number of leaked sites (Q) after treatment of mice with CM derived from parental HTB140 and LM1-744 cells (n = 9-11 mice). Scale bar 100m. All data represent the mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student’s t test).Supplemental Figure S8. NID1 promotes in vitro tube formation on matrigel. (A-B) Quantification and phase images of SCP28 (A), and HTB140 (B) vector and NID1-overexprssing cell lines. (C-D) Quantification and phase images of LM1a (C) and LM1-744 (D) control (vector and scrambled shRNA) and NID1 knockdown (KD1 and KD2) cell lines. (E) Quantification and phase images of MDA-MB-231 vector and NID1-overexpressing cell lines with and without antibody treatment against NID1 (anti-NID1). Scale bar 100?m. In vitro tube formation assays were performed at least in triplicate. Data represent the mean ± s.e.m. *P<0.05, **P<0.01, ****P<0.0001 (Student’s t test).Supplemental TablesSupplemental Table 1, as a separate Excel file. Mass spectrometry data. Supplemental Table 2, as a separate Excel file. Proteins in breast cancer Up-LMSS.Supplemental Table 3, as a separate Excel file. Proteins in breast cancer Down-LMSS.Supplemental Table 4, as a separate Excel file. Proteins in melanoma Up-LMSS.Supplemental Table 5, as a separate Excel file. Proteins in melanoma Down-LMSS.Supplemental Table 6, as a separate Excel file. GO enrichment analysis of significantly enriched Biological Processes in the breast cancer Up-LMSS.Supplemental Table 7, as a separate Excel file. GO enrichment analysis of significantly enriched Biological Processes in the melanoma Up-LMSS.Supplemental Table 8, as a separate Excel file. GO enrichment analysis of significantly enriched Molecular Functions in the breast cancer Up-LMSS.Supplemental Table 9, as a separate Excel file. GO enrichment analysis of significantly enriched Molecular Functions in the melanoma Up-LMSS. Supplemental Table 10, as a separate Excel file. Cell authentication results by Genetica STR profiling.Supplemental Table 11, as a separate Excel file. Melanoma TCGA clinical dataset used for survival analysis. Supplemental SoftwareSupplemental File 1, as a separate file. MATLAB code for TCGA melanoma patient survival analysis. Supplemental File 2, as a separate file. Text file of the MATLAB code for TCGA melanoma patient survival analysis. ................
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