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Supplemental Figure 1 Characterization of Lung Tumors In Supplemental Figure 1 Characterization of lung tumors in KC mice. (A) Scatter plots of visible spontaneous tumors counted on the lung surfaces at necropsy. Each dot represents a value from a single mouse, and the bars and whiskers show the mean SD for each cohort from a single experiment. P value, two-tailed Student’s t-test. (B) Histologic analysis of spontaneous lung tumors in (A). Results indicate the numbers of lung lesions per histologic subtype per genotype. N, cohort sample sizes. P values, Fisher's exact test. Supplemental Figure 2 Surfactant protein C (SPC) expression in p21-deficient (KC) and –replete (K) tumor cell lines. Immunofluorescence staining with anti-surfactant protein C antibody (green) or normal IgG as a negative control. DAPI, blue. SPC/DAPI and IgG/DAPI are merged images. Scale bar, 20 µm. Supplemental Figure 3 Somatic recombination of latent KrasG12D allele and detection of p21 protein in p21-deficient (KC) and –replete (K) tumor cell lines. (A) PCR analysis of the K-rasLA1 and K-rasWT alleles in genomic DNA. Somatic recombination of the K-rasLA1 allele leads to loss of the PCR product (upper band, arrow). Wild-type K-ras allele detected (250 bp). Tail DNAs from KrasLA1 mice (KrasLA1/+) and wild-type mice (Kras+/+) were used as positive and negative controls, respectively, for the presence of the germline K-rasLA1 allele. (B) Immunoblot analysis of p21 expression. Actin was used as a loading control. Supplemental Figure 4 Growth and metastasis of p21-deficient (KC) tumor cell lines in Cdkn1a-null or –wild-type mice. (A) Immunoblot analysis of KC3 cells stably transfected with p21 or empty expression vector (Vec). Actin was used as the loading control. (B and C) Scatter plots of primary tumor weights (B) and total numbers of lung metastases (C) from a single experiment in which mice with the indicated genotypes were injected subcutaneously with KC3 cells that express ectopic p21 or empty vector. Each dot represents a value from a single mouse, and the bars and whiskers show the mean SD. P values, two-tailed Student’s t-test. (D-G) Scatter plots of primary tumor weights (D and F) and total numbers of lung metastases (E and G) in mice with the indicated genotypes injected subcutaneously with KC2 cells. Each dot represents a value from a single mouse, and the bars and whiskers show the mean SD from a single experiment. P values, two-tailed Student’s t-test. Supplemental Figure 5 Proliferation and colony formation of p21-deficient (KC) and –replete (K) tumor cell lines (A) Proliferation assay (WST-1). Mean values ( SD) of triplicate samples from a single experiment expressed relative to d=1, which was set at 1.0. (B) Soft agar colony assay. Mean values ( SD) from a single experiment analyzed after 21 days in culture (bar graph). P values, two-tailed Student’s t-test. One-way ANOVA (P<0.0001). Supplemental Figure 6 In vitro properties of p21-deficient (KC) and –replete (K) tumor cell lines. (A) Invading K1 cells in mono-culture (Con) or co-culture with KC2 cells (KC2) in Boyden chambers (images). Scale bars, 200 μm. Mean SD of triplicate samples from a single experiment (bar graphs). (B) K1 cell spheroids mono-cultured (Con) or co-cultured with KC2 cells (KC2) in collagen gels. Invasive protrusions quantified 24 hours after seeding. Scale bars, 100 μm. Mean values ( SD) of triplicate samples from a single experiment (bar graph). (C) Relative cell density (WST-1 assays) of p21-transfected (p21) or vector-transfected (Vec) KC3 cells in monolayer culture. Mean SD of triplicate wells from a single experiment expressed relative to d = 1, which was set at 1.0. (D) Migrating and invading KC3 cells transfected with p21 or empty vector in Boyden chambers (images). Scale bars are 200 μm. Mean SD of cell numbers from triplicate wells in a single experiment (bar graphs). P values, two-tailed Student’s t-test. Supplemental Figure 7 Karyotypic and transcriptomic analyses of p21-deficient (KC) and –replete (K) tumor cell lines. (A) Heat map illustration of chromosome numbers determined by karyotypic analysis. Mean numbers of chromosomes per cell determined from 75-100 metaphases per cell line. (B) Spectral karyotypic analysis of KC4 cells. The arrow points to derivative chromosome (1)T(1H5;6A2). (C) Heat map representation of genes differentially expressed relative to KC4 cells in at least 1 of the 3 highly metastatic KC cells (KC1-3). Triplicate samples of each cell line represented by adjacent columns. Numbers of gene probes increased (yellow) or decreased (blue) relative to KC4 (black) in a single experiment indicated on right. (D) Heat map representation of data from (C) showing genes differentially expressed between highly (KC1-3) and poorly (KC4) metastatic cells. Number of gene probes increased (yellow) or decreased (blue) relative to KC4 (black) indicated on left. (E) Q-PCR verification of genes with the greatest fold-upregulation (red) or -downregulation (blue) from (D). Log2 ratio of the mean values of triplicate samples of highly (KC1-3) versus poorly (KC4) metastatic cells. P values, two-tailed Student’s t-test. Supplemental Figure 8 p21-independent regulation of LH2, AQP1, and TIMP3 expression. (A and B) Q-PCR analysis of p21-deficient (KC) and –replete (K) tumor cell lines (A) and KC3 cells stably transfected with p21 or empty (Vec) expression vector (B). Results from a single experiment expressed as mean ± SD values of triplicate samples. P values, two-tailed Student’s t-test. Supplemental Figure 9 AQP1- and TIMP3-independent regulation of cell migration and invasion. (A) Q-PCR analysis of KC2 cells stably transfected with AQP1 or TIMP3 cDNA expression vectors. Mean ± SD values of triplicate samples (bar graph). Results from a single experiment expressed relative to control transfectants, which were set at 1.0. P values, two-tailed Student’s t-test. (B) Migrating and invading KC2 cell transfectants from (A) in Boyden chambers (images). Scale bars, 200 μm. Mean ± SD values of triplicate wells from a single experiment (bar graph). Supplemental Figure 10 LH2 promotes tumor cell migration and invasion but not proliferation. (A) Q-PCR analysis (bar graph) and immunoblot analysis (gels) of KC1 cells stably transfected with vectors expressing LH2 shRNA (SH1, SH3, and SH5) or scrambled control shRNA (SCR). Q-PCR results are expressed as mean ± SD values of triplicate samples from a single experiment. Results expressed relative to control transfectants, which were set at 100%. Actin was used as loading control. (B) Migrating and invading KC1 cell transfectants in Boyden chambers (images). Scale bars, 200 μm. Mean ± SD of triplicate wells from a single experiment (bar graphs). Results expressed relative to control transfectants, which were set at 100%. (C) Proliferation assay (WST-1) on KC2 cells transfected with LH2-specific shRNAs (SH1, SH2, or SH5) or control shRNAs (SCR). Mean values ( SD) of triplicate samples from a single experiment expressed relative to d=1, which was set at 1.0. (D) Soft agar colony assay after 21 days in culture. Mean values ( SD) of triplicate samples from a single experiment. P values, two-tailed Student’s t- test. Supplemental Figure 11 LH2 promotes the migration and invasion of human A549 lung cancer cells. (A) Q-PCR analysis of LH2 expression in a panel of 12 human lung cancer cell lines. Normalized Q-PCR results are expressed as mean ± SD of triplicate samples from a single experiment. (B) Immunoblot analysis of A549 cells stably transfected with 1 of 2 LH2 shRNAs (SHC or SHD) or control shRNA (FF2). Actin used as loading control. (C) Migrating and invading A549 cell transfectants in Boyden chambers (images). Scale bars, 200 μm. Mean ± SD of triplicate wells from a single experiment (bar graphs). Results expressed relative to control transfectants, which were set at 100%. P values, two-tailed Student’s t-test. Supplemental Figure 12 No differences in MMP2 activity between KC cell lines. Gelatinase zymography of conditioned media samples from a single experiment on the indicated cells. Bands correspond with pro- and cleaved forms of MMP2. Supplemental Figure 13 Differential expression of MMP family members between KC cell lines. Q-PCR analysis of mRNA levels expressed as mean ± SD values of triplicate samples from a single experiment. P values indicate comparisons between poorly (KC4) and highly (KC1-3) metastatic cell lines (two-tailed Student’s t-test). Supplemental Figure 14 Differential expression of integrin family members between KC cell lines. Q-PCR analysis of mRNA levels expressed as mean ± SD values of triplicate samples from a single experiment. P values indicate comparisons between poorly (KC4) and highly (KC1-3) metastatic cell lines (two-tailed Student’s t-test). Supplemental Figure 15 Confirmation that highly and poorly metastatic KC cells have expected relative LH2 levels, tumor sizes, and metastatic activities prior to performing collagen cross-link analysis. (A) Q- PCR analysis of K1, KC2, and KC4 cells. Results expressed as mean ± SD values of triplicate samples from a single experiment. P values, two-tailed Student’s t-test and one-way ANOVA (P<0.0001). (B) Scatter plots of primary tumor weights (left panel) and total numbers of lung metastases (right panel) in Cdkn1a-null mice injected subcutaneously with indicated cells. Each dot represents a value from a single mouse, and the bars and whiskers show the mean SD from a single experiment. P values, two-tailed Student’s t-test. One-way ANOVA (P<0.01). Supplemental Figure 16 Up-regulation of LOX levels in KC4 cells. Q-PCR analysis of the indicated mRNAs in KC1-4 cells. Results are expressed as mean ± SD values of triplicate samples from a single experiment.
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