Fig 2: COPII insufficiency affects secretion of ECM proteins, but restoration of only SAR1A and SEC24D does not appear to adversely affect growth in orthotopic xenograft model(A) SAR1A and SEC24D mRNA transcripts from 143B.ST and143B.ST(+COPII) cells were analyzed by qRT-PCR and expressed relative to their values in hFOBl.19 cells (n = 3). The differences in values for 143B.ST and the osteoblast line were significant to p =0 .001 for SAR1A (***) and p =0 .0001 for SEC24D (****). The restored values were greater than that of the hFOB1.19 for SAR1A p = 0.01 (**) and slightly less for SEC24D p = 0.05 (*). Error bars indicate mean ± SD.(B) Spectral counts were transformed to log2 and averaged (n = 2 biological replicates) and fold change calculated. Fold change values were plotted against the -log10 of the p value with all significant hits of p = 0.01 indicated (red). The p values were derived by G-test.(C) Uniprot IDs of significantly, differentially expressed proteins were analyzed using QuickGO for subcellular location determination.(D) Cells were cultured in serum free media for 20 h and secreted proteins were TCA-precipitated, followed by Western blot analysis with anti-TIMP1 and anti-vimentin.(E) 100K cells of 143B.ST or 143B(+COPII) were implanted into the right tibia of CD1-nude mice (n = 10 for each line). On day 3 postimplantation, the luciferase signal was measured and then every 7 days thereafter to monitor tumor growth. Total bioluminescence flux of the region of interest (right tibia) was plotted against days postimplant.(F) Caliper measurements were recorded to supplement luciferase imaging, and tumor volume calculated.Statistical differences between both the luciferase signal and tumor volume for 143B.ST and 143B.ST(+COPII) cells were computed using unpaired, two-tailed Student’s t-tests for each time point comparison and group overall and were discovered to be nonsignificant. All error bars indicate mean ± SD.

Fig 3: OS, PDXs, and cell lines have reduced levels of a subset of COPII vesicle components(A) RNA-Seq analyses of the indicated COPII component transcripts from two SJCRH PDX tumors (solid bars) normalized to normal human osteoblast tissue samples. RNA seq data from the indicated OS cell lines (striped bars) were averaged and normalized to hFOB1.1 9 (n = 3 biological replicates). **P=0.01, ***P=0.001 and ****P=0.0001(B) Cell lysates from the indicated cell lines were examined by western blot analyses for SAR1A and SEC24D expression. HSC70 or GAPDH are included as loading controls.(C) SaOS2 cells were transfected with 1 µg of SEC24D and SAR1A expression constructs either individually or in combination. 48 hrs post-transfection cells were metabolically labeled for 16 h. Both cell lysates (L) and culture supernatant (M) were immunoprecipitated with anti-collagen-I serum (anti-SP1.D8) and analyzed by reduced SDS-PAGE. The mobility of procollagen monomers (?) in the cell lysates is labeled, as are collagen trimers (*) and dimers (**) in the media.(D) The scatter plot displays the log2 fold change of six COPII transcripts from 108 OS patient tumor samples compared to normal osteoblast tissue. The (black) midline bar indicates the mean value for the tumor samples.