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Charles River Laboratories tc 71 cells
$ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots <t>of</t> <t>TC-71</t> lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.$
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1) Product Images from "ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis"

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

Journal: Cancer Research Communications

doi: 10.1158/2767-9764.CRC-26-0068

$ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots of TC-71 lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.$
Figure Legend Snippet: ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots of TC-71 lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.

Techniques Used: Western Blot, Transfection, CRISPR, Knock-Out, Immunoprecipitation, Staining, Recombinant, Bioprocessing, Binding Assay, Injection, Sequencing, Phospho-proteomics, Glycoproteomics, In Silico, Comparison, Control, Clinical Proteomics, Membrane

Genome-wide CRISPR/Cas9 transcriptional activation screen reveals novel prometastatic genes in Ewing sarcoma. A, Schematic overview of the in vivo CRISPR/Cas9 genome-wide transcriptional activation screening experiment (TSS, transcription start site). B, Four Ewing sarcoma cell lines were injected into 48 hpf zebrafish embryos at three different cell densities ( n = 12 for each condition) and the number of fish with metastasis to the tail was scored every day for 5 days. C, Read counts per million for each gRNA detected from the genome-wide CRISPR/Cas9 transcriptional activation screening experiment. The eight genes chosen for further study are indicated in bold. D, Eight candidate genes from the screen were stably overexpressed in TC-71 cells and tested in zebrafish xenograft assays (*, P < 0.05; **, P < 0.01; χ 2 test). The red line indicates the baseline intravasation of TC-71 cells transfected with EV. The means and SEMs are shown. The dots represent individual experiments and the total number of fish in all the experiments combined is provided above each condition. E, TC-71 cells stably overexpressing eight different genes were tested via a scratch assay. The dots indicate independent experiments. The means and SEMs are shown (*, P < 0.05, Student t test). The red line indicates the average percent wound closure of TC-71 cells transfected with EV. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/pw6o2c1 .]

Figure Legend Snippet: Genome-wide CRISPR/Cas9 transcriptional activation screen reveals novel prometastatic genes in Ewing sarcoma. A, Schematic overview of the in vivo CRISPR/Cas9 genome-wide transcriptional activation screening experiment (TSS, transcription start site). B, Four Ewing sarcoma cell lines were injected into 48 hpf zebrafish embryos at three different cell densities ( n = 12 for each condition) and the number of fish with metastasis to the tail was scored every day for 5 days. C, Read counts per million for each gRNA detected from the genome-wide CRISPR/Cas9 transcriptional activation screening experiment. The eight genes chosen for further study are indicated in bold. D, Eight candidate genes from the screen were stably overexpressed in TC-71 cells and tested in zebrafish xenograft assays (*, P < 0.05; **, P < 0.01; χ 2 test). The red line indicates the baseline intravasation of TC-71 cells transfected with EV. The means and SEMs are shown. The dots represent individual experiments and the total number of fish in all the experiments combined is provided above each condition. E, TC-71 cells stably overexpressing eight different genes were tested via a scratch assay. The dots indicate independent experiments. The means and SEMs are shown (*, P < 0.05, Student t test). The red line indicates the average percent wound closure of TC-71 cells transfected with EV. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/pw6o2c1 .]

Techniques Used: Genome Wide, CRISPR, Activation Assay, In Vivo, Injection, Stable Transfection, Transfection, Wound Healing Assay

ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Figure Legend Snippet: ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Techniques Used: Binding Assay, Recombinant, Injection, Expressing, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control

ROME expression drives metastasis development in vivo . A, Timeline of tail vein metastasis experiments. B, In a tail vein metastasis model, mice injected with ROME-overexpressing TC-71 cells ( n = 11) had a higher total mass of metastatic nodules than did controls ( n = 10). C, In a tail vein metastasis model, mice injected with ROME-KO TC-71 cells ( n = 11) had a lower total mass of metastases than controls injected with WT TC71 cells ( n = 11). Statistical significance was determined via an unpaired t test (one-tailed) for B and C . D, Diagram of the orthotopic Ewing sarcoma leg amputation experimental timeline. E, Representative gross images of lungs from mice injected with TC-71 cells overexpressing ROME or with EV (left) with quantification (middle). Representative images of H&E staining are shown on the right confirming metastatic nodules in the lungs of SCID mice. Statistical significance was calculated by the Fisher exact test (one-sided). F, TC-71 ROME-KO cells displayed significantly reduced primary tumor recurrence [ n = 10 per group, log-rank (Mantel–Cox) test]. G, ROME is expressed throughout many different tumor types (CNS, central nervous system; HEPAC, hepatocellular carcinoma and cholangioma; KICH, kidney chromophobe and other renal carcinoma; KIPCC, papillary cell renal carcinoma; OV, ovarian cystadenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; SCC/BLCA, squamous cell or bladder carcinoma; TGCT SEM, testicular germ cell tumor, seminoma). H, Kaplan–Meier curves of patient survival stratified by high (red line) and low (black line) ROME expression (from the Kaplan–Meier plotter database). [ A and D, Created in BioRender. Lab, S. (2026) https://BioRender.com/atgnfw0 .]

Figure Legend Snippet: ROME expression drives metastasis development in vivo . A, Timeline of tail vein metastasis experiments. B, In a tail vein metastasis model, mice injected with ROME-overexpressing TC-71 cells ( n = 11) had a higher total mass of metastatic nodules than did controls ( n = 10). C, In a tail vein metastasis model, mice injected with ROME-KO TC-71 cells ( n = 11) had a lower total mass of metastases than controls injected with WT TC71 cells ( n = 11). Statistical significance was determined via an unpaired t test (one-tailed) for B and C . D, Diagram of the orthotopic Ewing sarcoma leg amputation experimental timeline. E, Representative gross images of lungs from mice injected with TC-71 cells overexpressing ROME or with EV (left) with quantification (middle). Representative images of H&E staining are shown on the right confirming metastatic nodules in the lungs of SCID mice. Statistical significance was calculated by the Fisher exact test (one-sided). F, TC-71 ROME-KO cells displayed significantly reduced primary tumor recurrence [ n = 10 per group, log-rank (Mantel–Cox) test]. G, ROME is expressed throughout many different tumor types (CNS, central nervous system; HEPAC, hepatocellular carcinoma and cholangioma; KICH, kidney chromophobe and other renal carcinoma; KIPCC, papillary cell renal carcinoma; OV, ovarian cystadenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; SCC/BLCA, squamous cell or bladder carcinoma; TGCT SEM, testicular germ cell tumor, seminoma). H, Kaplan–Meier curves of patient survival stratified by high (red line) and low (black line) ROME expression (from the Kaplan–Meier plotter database). [ A and D, Created in BioRender. Lab, S. (2026) https://BioRender.com/atgnfw0 .]

Techniques Used: Expressing, In Vivo, Injection, One-tailed Test, Staining

ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 ( A ) and TC-32 ( B ) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D . E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca 2+ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca 2+ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L .

Figure Legend Snippet: ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 ( A ) and TC-32 ( B ) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D . E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca 2+ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca 2+ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L .

Techniques Used: Control, Stable Transfection, Recombinant, Luciferase, Knockdown, Activity Assay, Expressing, Western Blot, Two Tailed Test, Binding Assay, Injection, Co-Immunoprecipitation Assay, Transfection

ROME expression increases cell motility and chemotaxis in vitro . A, TC-71 cells stably overexpressing ROME demonstrated increased wound closure compared with that of a control cell line, and TC-71 cells with ROME KO by CRISPR/Cas9 demonstrated decreased wound closure compared with that of WT cells in scratch assays. At least two independent experiments were performed, and average wound closure with SEM was quantified. Statistical significance was calculated by an unpaired t test (two-tailed). B–E, Compared with EV control cells, A4573, TC-71, and Hs746.T cells stably expressing ROME presented increased chemotaxis in Boyden chamber assays. F, CHLA-10 cells with ROME KO via CRISPR/Cas9 showed decreased chemotaxis compared with that of WT cells. G–I, ROME KO (TC-71) or knockdown (769-P and Hs746.T) decreased the chemotaxis of cancer cells. J and K, Rescue of ROME expression by an exogenous vector restored chemotaxis in both CHLA-10 and TC-71 cells. At least two independent experiments with three biological replicates were performed for each Boyden chamber assay. Statistical significance was calculated by an unpaired t test (two-tailed).

Figure Legend Snippet: ROME expression increases cell motility and chemotaxis in vitro . A, TC-71 cells stably overexpressing ROME demonstrated increased wound closure compared with that of a control cell line, and TC-71 cells with ROME KO by CRISPR/Cas9 demonstrated decreased wound closure compared with that of WT cells in scratch assays. At least two independent experiments were performed, and average wound closure with SEM was quantified. Statistical significance was calculated by an unpaired t test (two-tailed). B–E, Compared with EV control cells, A4573, TC-71, and Hs746.T cells stably expressing ROME presented increased chemotaxis in Boyden chamber assays. F, CHLA-10 cells with ROME KO via CRISPR/Cas9 showed decreased chemotaxis compared with that of WT cells. G–I, ROME KO (TC-71) or knockdown (769-P and Hs746.T) decreased the chemotaxis of cancer cells. J and K, Rescue of ROME expression by an exogenous vector restored chemotaxis in both CHLA-10 and TC-71 cells. At least two independent experiments with three biological replicates were performed for each Boyden chamber assay. Statistical significance was calculated by an unpaired t test (two-tailed).

Techniques Used: Expressing, Chemotaxis Assay, In Vitro, Stable Transfection, Control, CRISPR, Two Tailed Test, Knockdown, Plasmid Preparation, Boyden Chamber Assay

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Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME is a 153–amino acid transmembrane glycoprotein. A, Western blots of TC-71 lysates transfected with EV or ROME-HA for 24 hours blotted with the commercially available anti-HA antibody, ICD-6 (monoclonal antibody against the predicted ICD of ROME), and ECD-5 (monoclonal antibody against the predicted ECD of ROME) are shown. B, ROME was knocked out via CRISPR/Cas9 in TC-71 cells. Successful knockout was confirmed by immunoprecipitation and Western blotting with a ROME monoclonal antibody (ICD-6). C, Coomassie blue staining was used to verify the purity of recombinant ROME-FL and ECD proteins and the synthetic ICD peptide. Western blotting with ROME ICD-6 and ECD-5 monoclonal antibodies was performed to confirm protein identities. D, SPR sensorgrams demonstrating the binding of the anti-ROME monoclonal antibody ICD-6 to the ROME ICD and FL proteins and the binding of the anti-ROME monoclonal antibody ECD-5 to the ROME ECD and FL proteins. The antibodies were injected in duplicate at 200, 100, 50, 25, 12.5, and 6.25 nmol/L. The red lines are the actual data and the black lines indicate the curve fit. E, Three-dimensional structure of ROME predicted by AlphaFold. F, A diagram of the ROME amino acid sequence with three predicted domains is shown. Circles correspond to phosphorylation sites and triangles correspond to glycosylation sites. The black color designates sites predicted in silico and the blue color designates sites confirmed by MS. G, Homology between human, mouse, and zebrafish ROME proteins is shown. The Drosophila inaF motif (from the INAF-D protein) is shown for comparison. The black line box indicates the transmembrane domain. Asterisks (*) indicate identical amino acids (aa) and colons (:) indicate amino acids with similarity among all three species. H, Phylogenetic tree showing the high evolutionary conservation of the ROME protein in vertebrates. I, Compared with the eGFP control, ROME-GFP localizes to the plasma membrane of TC-71 cells (quantification shown in the graph to the right). J, ROME-HA was expressed in TC-71 cells and detected primarily in the membrane fraction, as well as the soluble nuclear fraction. α-Tubulin was used as a cytoplasmic fraction control, CD99 was used as a membrane fraction control, and lamin A/C was used as a soluble nuclear fraction control. K, Lysates from TC-71 cells transfected with EV or ROME-FLAG were treated with a deglycosylase mixture. Western blotting with anti-FLAG antibody was performed to detect ROME-FLAG.

Article Snippet: For the tail vein experiments, 1 million TC-71 cells (WT and ROME KO or stably expressing ROME cDNA versus an EV control cell line) were injected into the tail vein of 6-week-old female SCID beige mice (Charles River Laboratories, RRID: IMSR_CRL:250).

Techniques: Western Blot, Transfection, CRISPR, Knock-Out, Immunoprecipitation, Staining, Recombinant, Bioprocessing, Binding Assay, Injection, Sequencing, Phospho-proteomics, Glycoproteomics, In Silico, Comparison, Control, Clinical Proteomics, Membrane

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: Genome-wide CRISPR/Cas9 transcriptional activation screen reveals novel prometastatic genes in Ewing sarcoma. A, Schematic overview of the in vivo CRISPR/Cas9 genome-wide transcriptional activation screening experiment (TSS, transcription start site). B, Four Ewing sarcoma cell lines were injected into 48 hpf zebrafish embryos at three different cell densities ( n = 12 for each condition) and the number of fish with metastasis to the tail was scored every day for 5 days. C, Read counts per million for each gRNA detected from the genome-wide CRISPR/Cas9 transcriptional activation screening experiment. The eight genes chosen for further study are indicated in bold. D, Eight candidate genes from the screen were stably overexpressed in TC-71 cells and tested in zebrafish xenograft assays (*, P < 0.05; **, P < 0.01; χ 2 test). The red line indicates the baseline intravasation of TC-71 cells transfected with EV. The means and SEMs are shown. The dots represent individual experiments and the total number of fish in all the experiments combined is provided above each condition. E, TC-71 cells stably overexpressing eight different genes were tested via a scratch assay. The dots indicate independent experiments. The means and SEMs are shown (*, P < 0.05, Student t test). The red line indicates the average percent wound closure of TC-71 cells transfected with EV. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/pw6o2c1 .]

Techniques: Genome Wide, CRISPR, Activation Assay, In Vivo, Injection, Stable Transfection, Transfection, Wound Healing Assay

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME drives intravasation through vimentin. A, Timeline of zebrafish xenograft experiments. B, Zebrafish xenograft experiments with three different ROME-overexpressing Ewing sarcoma cell lines. Statistical significance was determined via the Fisher exact test (two-sided). C, Zebrafish xenograft experiments comparing WT and ROME-KO TC-71 cells. Statistical significance was determined via the Fisher exact test (one-sided). D, ROME and vimentin proteins co-immunoprecipitate in three different Ewing sarcoma cell lines. For TC-32 ROME blot, line denotes higher exposure on left to visualize input and lower exposure on right. E, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and vimentin protein. Vimentin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 2,500, 800, 280, 90, 30, and 10 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. F, ROME expression reduces vimentin phosphorylation at serine 56 (S56) in a tet-inducible ROME-HA expression system (DOX = doxycycline 250 ng/mL). Densitometry analysis of Western blot bands is shown in the bottom right corner ( n = 4 independent experiments). G, Western blotting was used to confirm vimentin knockdown by siRNA in STA-ET-7.2 cells with EV or ROME overexpression. H, Vimentin knockdown selectively reduced ROME-driven intravasation in ROME-overexpressing STA-ET-7.2 cells compared with an EV control [ n = 102 (EV + siNT), 116 (ROME + siNT), 88 (EV + siVimentin), and 119 (ROME + siVimentin)]. I, ROME mRNA expression positively correlates with vimentin mRNA expression in analysis of 13,313 patient samples. [ A, Created in BioRender. Lab, S. (2026) https://BioRender.com/fp8c676 .]

Techniques: Binding Assay, Recombinant, Injection, Expressing, Phospho-proteomics, Western Blot, Knockdown, Over Expression, Control

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME expression drives metastasis development in vivo . A, Timeline of tail vein metastasis experiments. B, In a tail vein metastasis model, mice injected with ROME-overexpressing TC-71 cells ( n = 11) had a higher total mass of metastatic nodules than did controls ( n = 10). C, In a tail vein metastasis model, mice injected with ROME-KO TC-71 cells ( n = 11) had a lower total mass of metastases than controls injected with WT TC71 cells ( n = 11). Statistical significance was determined via an unpaired t test (one-tailed) for B and C . D, Diagram of the orthotopic Ewing sarcoma leg amputation experimental timeline. E, Representative gross images of lungs from mice injected with TC-71 cells overexpressing ROME or with EV (left) with quantification (middle). Representative images of H&E staining are shown on the right confirming metastatic nodules in the lungs of SCID mice. Statistical significance was calculated by the Fisher exact test (one-sided). F, TC-71 ROME-KO cells displayed significantly reduced primary tumor recurrence [ n = 10 per group, log-rank (Mantel–Cox) test]. G, ROME is expressed throughout many different tumor types (CNS, central nervous system; HEPAC, hepatocellular carcinoma and cholangioma; KICH, kidney chromophobe and other renal carcinoma; KIPCC, papillary cell renal carcinoma; OV, ovarian cystadenocarcinoma; PAAD, pancreatic ductal adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; SCC/BLCA, squamous cell or bladder carcinoma; TGCT SEM, testicular germ cell tumor, seminoma). H, Kaplan–Meier curves of patient survival stratified by high (red line) and low (black line) ROME expression (from the Kaplan–Meier plotter database). [ A and D, Created in BioRender. Lab, S. (2026) https://BioRender.com/atgnfw0 .]

Techniques: Expressing, In Vivo, Injection, One-tailed Test, Staining

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME protein negatively regulates the Wnt pathway and calcium signaling in human cancer cells. A and B, Compared with EV control cells, TC-71 ( A ) and TC-32 ( B ) cells stably overexpressing ROME were less responsive to recombinant human Wnt3a protein as measured by TOPFlash β-catenin–responsive luciferase reporter. C, TC-71 cells with ROME knockdown by siRNA were more responsive to Wnt3a. D, Endogenous Wnt pathway activity in the colorectal cancer cell line HCT116 was significantly decreased by ROME expression (Western blots confirming ROME-HA expression on the right). Statistical significance was calculated by an unpaired t test (two-tailed) for A–D . E, ROME co-immunoprecipitates with β-catenin in β-catenin pulldown experiments. F, β-Catenin co-immunoprecipitates with ROME in ROME pulldown experiments. G, SPR sensorgrams showing direct binding between the recombinant full-length ROME protein and β-catenin protein. β-Catenin was immobilized on the surface and full-length ROME protein was injected in duplicate at concentrations of 1,250, 416.7, 138.9, 46.3, 15.4, and 5.12 nmol/L. The red lines are the actual data, and the black lines indicate the curve fit. H, ROME expression decreases β-catenin and TCF4 interaction (measured by co-IP). I–K, The Ca 2+ response was decreased in A4573, STA-ET-7.2, and TC-71 cells overexpressing ROME compared with that in EV-transfected cells when the cells were stimulated with ATP or FBS after overnight serum starvation. L, The Ca 2+ response was greater in the TC-71 cells with ROME KO than in the WT cells. Statistical significance was calculated by an unpaired t test (two-tailed) for I–L .

Techniques: Control, Stable Transfection, Recombinant, Luciferase, Knockdown, Activity Assay, Expressing, Western Blot, Two Tailed Test, Binding Assay, Injection, Co-Immunoprecipitation Assay, Transfection

Journal: Cancer Research Communications

Article Title: ROME, an Ancient Gene with a Novel Function in Vertebrates, Is a Key Modulator of Embryonal Development and Cancer Metastasis

doi: 10.1158/2767-9764.CRC-26-0068

Figure Lengend Snippet: ROME expression increases cell motility and chemotaxis in vitro . A, TC-71 cells stably overexpressing ROME demonstrated increased wound closure compared with that of a control cell line, and TC-71 cells with ROME KO by CRISPR/Cas9 demonstrated decreased wound closure compared with that of WT cells in scratch assays. At least two independent experiments were performed, and average wound closure with SEM was quantified. Statistical significance was calculated by an unpaired t test (two-tailed). B–E, Compared with EV control cells, A4573, TC-71, and Hs746.T cells stably expressing ROME presented increased chemotaxis in Boyden chamber assays. F, CHLA-10 cells with ROME KO via CRISPR/Cas9 showed decreased chemotaxis compared with that of WT cells. G–I, ROME KO (TC-71) or knockdown (769-P and Hs746.T) decreased the chemotaxis of cancer cells. J and K, Rescue of ROME expression by an exogenous vector restored chemotaxis in both CHLA-10 and TC-71 cells. At least two independent experiments with three biological replicates were performed for each Boyden chamber assay. Statistical significance was calculated by an unpaired t test (two-tailed).

Techniques: Expressing, Chemotaxis Assay, In Vitro, Stable Transfection, Control, CRISPR, Two Tailed Test, Knockdown, Plasmid Preparation, Boyden Chamber Assay

Kaplan-Meier survival analysis and differential expression of IMPDH2 in sarcoma. (A) Overall survival curve based on IMPDH2 expression in patients with sarcoma from the TCGA cohort (n = 262). Patients were stratified into low IMPDH2 expression (blue line) and high IMPDH2 expression (red line) groups. Kaplan-Meier curves show that higher expression of IMPDH2 is associated with a trend towards reduced survival, although this did not reach statistical significance (log-rank p = 0.061). The hazard ratio for high expression was 1.5, indicating a 50% increase in the risk of death for patients with high expression of IMPDH2 ( p = 0.063). (B) Differential expression of IMPDH2 between sarcoma (Tumor, n = 262) and normal tissues (Normal, n = 2) from the TCGA dataset. IMPDH2 expression, measured in transcripts per million (TPM), is significantly higher in tumor samples than in normal tissues. (C) Western blot analysis to detect expression of IMPDH2 in Ewing's sarcoma cell lines (RD-ES, SK-ES-1, and TC71). HeLa (human cervical carcinoma cell line) and MCF-7 (human breast cancer cell line) cells were used as positive controls. α/β-tubulin served as a loading control.

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Kaplan-Meier survival analysis and differential expression of IMPDH2 in sarcoma. (A) Overall survival curve based on IMPDH2 expression in patients with sarcoma from the TCGA cohort (n = 262). Patients were stratified into low IMPDH2 expression (blue line) and high IMPDH2 expression (red line) groups. Kaplan-Meier curves show that higher expression of IMPDH2 is associated with a trend towards reduced survival, although this did not reach statistical significance (log-rank p = 0.061). The hazard ratio for high expression was 1.5, indicating a 50% increase in the risk of death for patients with high expression of IMPDH2 ( p = 0.063). (B) Differential expression of IMPDH2 between sarcoma (Tumor, n = 262) and normal tissues (Normal, n = 2) from the TCGA dataset. IMPDH2 expression, measured in transcripts per million (TPM), is significantly higher in tumor samples than in normal tissues. (C) Western blot analysis to detect expression of IMPDH2 in Ewing's sarcoma cell lines (RD-ES, SK-ES-1, and TC71). HeLa (human cervical carcinoma cell line) and MCF-7 (human breast cancer cell line) cells were used as positive controls. α/β-tubulin served as a loading control.

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Quantitative Proteomics, Expressing, Western Blot, Control

Effects of AVN944 on growth and colony formation by TC71 Ewing's sarcoma cells. (A) Representative images of TC71 cells treated with 1 μM AVN944 or control (vehicle) over 5 days. Morphological changes were observed, with AVN944-treated cells showing lower cell density than control cells. Scale bar, 100 μm. (B) Growth curve for TC71 cells treated with 1 μM AVN944 (orange) or control (blue) for 5 days. AVN944 inhibited cell proliferation significantly when compared with the control. Data represent the mean ± SD of three independent experiments (n = 3, ** p < 0.01). (C) Representative images from a BrdU incorporation assay in which TC71 cells were treated with AVN944 for 3 h on Day 1. BrdU-positive cells (green) are actively proliferating. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) Quantification of BrdU-positive TC71 cells treated (or not) with AVN944. AVN944 reduced BrdU incorporation significantly, indicating a reduction in DNA synthesis (n = 3, ** p <0.01). (E) MTT assay of TC71 cells treated with AVN944. AVN944 treatment resulted in a significant reduction in cell viability compared with the control (n = 8, ** p <0.01). (F) Colony formation assay in which TC71 cells were treated with increasing concentrations of AVN944 (0-1 μM). Colonies were stained with crystal violet, which revealed dose-dependent inhibition of colony formation. (G) Quantification of colony formation in F. The number of colonies was counted using ImageJ software, and presented as a percentage of the control value. AVN944 reduced colony formation significantly and in a dose-dependent manner (* p < 0.05, ** p < 0.01 vs. control). Data represent the mean ± SD of three independent experiments (n = 3). (H) Measurement of intracellular ATP levels in TC71 cells treated with AVN944 for 24 h. AVN944 reduced cellular ATP levels significantly compared with the control, indicating a decline in cellular metabolic activity (** p < 0.01 vs. control). Data represent the mean ± SD of eight independent experiments (n = 8).

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Effects of AVN944 on growth and colony formation by TC71 Ewing's sarcoma cells. (A) Representative images of TC71 cells treated with 1 μM AVN944 or control (vehicle) over 5 days. Morphological changes were observed, with AVN944-treated cells showing lower cell density than control cells. Scale bar, 100 μm. (B) Growth curve for TC71 cells treated with 1 μM AVN944 (orange) or control (blue) for 5 days. AVN944 inhibited cell proliferation significantly when compared with the control. Data represent the mean ± SD of three independent experiments (n = 3, ** p < 0.01). (C) Representative images from a BrdU incorporation assay in which TC71 cells were treated with AVN944 for 3 h on Day 1. BrdU-positive cells (green) are actively proliferating. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (D) Quantification of BrdU-positive TC71 cells treated (or not) with AVN944. AVN944 reduced BrdU incorporation significantly, indicating a reduction in DNA synthesis (n = 3, ** p <0.01). (E) MTT assay of TC71 cells treated with AVN944. AVN944 treatment resulted in a significant reduction in cell viability compared with the control (n = 8, ** p <0.01). (F) Colony formation assay in which TC71 cells were treated with increasing concentrations of AVN944 (0-1 μM). Colonies were stained with crystal violet, which revealed dose-dependent inhibition of colony formation. (G) Quantification of colony formation in F. The number of colonies was counted using ImageJ software, and presented as a percentage of the control value. AVN944 reduced colony formation significantly and in a dose-dependent manner (* p < 0.05, ** p < 0.01 vs. control). Data represent the mean ± SD of three independent experiments (n = 3). (H) Measurement of intracellular ATP levels in TC71 cells treated with AVN944 for 24 h. AVN944 reduced cellular ATP levels significantly compared with the control, indicating a decline in cellular metabolic activity (** p < 0.01 vs. control). Data represent the mean ± SD of eight independent experiments (n = 8).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Control, BrdU Incorporation Assay, Staining, DNA Synthesis, MTT Assay, Colony Assay, Inhibition, Software, Activity Assay

Dose-dependent inhibition of TC71 cell proliferation by AVN944, and determination of the IC 50 . (A) Representative phase-contrast images of TC71 cells treated with varying concentrations of AVN944 (0-1 μM) for 3 days. Cells were imaged on Days 1, 2, and 3 post-treatment. AVN944 caused a concentration-dependent reduction in cell density, as well as changes in cell morphology, particularly at higher concentrations. Scale bar, 100 μm. (B) Dose-response curve for TC71 cells treated with AVN944 for 3 days. Cell viability was measured, and the half-maximal inhibitory concentration (IC 50 ) was calculated as 0.0535 μM, with an R² value of 0.998, indicating a solid fit of the data to the model. Data are presented as the mean ± SD of three independent experiments (n = 3).

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Dose-dependent inhibition of TC71 cell proliferation by AVN944, and determination of the IC 50 . (A) Representative phase-contrast images of TC71 cells treated with varying concentrations of AVN944 (0-1 μM) for 3 days. Cells were imaged on Days 1, 2, and 3 post-treatment. AVN944 caused a concentration-dependent reduction in cell density, as well as changes in cell morphology, particularly at higher concentrations. Scale bar, 100 μm. (B) Dose-response curve for TC71 cells treated with AVN944 for 3 days. Cell viability was measured, and the half-maximal inhibitory concentration (IC 50 ) was calculated as 0.0535 μM, with an R² value of 0.998, indicating a solid fit of the data to the model. Data are presented as the mean ± SD of three independent experiments (n = 3).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Inhibition, Concentration Assay

Time-dependent induction of apoptosis of TC71 Ewing's Sarcoma cells by. AVN944. (A) Flow cytometry analysis of DNA content in PI-stained TC71 cells treated with 5 μM AVN944 for 0, 48, 72, and 96 h. The sub-G1 (M1), G1 (M2), S phase (M3), and G2/M phase (M4) populations are indicated, along with a time-dependent increase in the sub-G1 population, indicative of apoptotic cells. (B) Measurement of the sub-G1 population at 48, 72, and 96 h post-treatment with AVN944. Data are presented as the mean ± SD from three independent experiments (n = 3, ** p < 0.01). (C) Flow cytometry analysis of apoptosis (using Annexin V and PI staining) in TC71 cells treated with 5 μM AVN944 for the indicated times. Annexin V-positive cells exhibit early-stage apoptosis, while PI-positive/Annexin V-negative cells are necrotic. An apparent time-dependent increase in the percentage of Annexin V-positive cells indicates progression from early apoptosis at 48 h to advanced apoptosis by 72 and 96 h. (D) Quantification of Annexin V-positive apoptotic cells over time. A time-dependent increase in Annexin V-positive cells was observed, with significant elevations at 48, 72, and 96 h, indicating progression of apoptosis. By 96 h, more than 78% of TC71 cells were Annexin V-positive, confirming marked induction of apoptosis (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3). (E) Percentage of viable cells over time post-treatment with AVN944. Viable cell populations decreased significantly at 48, 72, and 96 h (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3).

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: Time-dependent induction of apoptosis of TC71 Ewing's Sarcoma cells by. AVN944. (A) Flow cytometry analysis of DNA content in PI-stained TC71 cells treated with 5 μM AVN944 for 0, 48, 72, and 96 h. The sub-G1 (M1), G1 (M2), S phase (M3), and G2/M phase (M4) populations are indicated, along with a time-dependent increase in the sub-G1 population, indicative of apoptotic cells. (B) Measurement of the sub-G1 population at 48, 72, and 96 h post-treatment with AVN944. Data are presented as the mean ± SD from three independent experiments (n = 3, ** p < 0.01). (C) Flow cytometry analysis of apoptosis (using Annexin V and PI staining) in TC71 cells treated with 5 μM AVN944 for the indicated times. Annexin V-positive cells exhibit early-stage apoptosis, while PI-positive/Annexin V-negative cells are necrotic. An apparent time-dependent increase in the percentage of Annexin V-positive cells indicates progression from early apoptosis at 48 h to advanced apoptosis by 72 and 96 h. (D) Quantification of Annexin V-positive apoptotic cells over time. A time-dependent increase in Annexin V-positive cells was observed, with significant elevations at 48, 72, and 96 h, indicating progression of apoptosis. By 96 h, more than 78% of TC71 cells were Annexin V-positive, confirming marked induction of apoptosis (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3). (E) Percentage of viable cells over time post-treatment with AVN944. Viable cell populations decreased significantly at 48, 72, and 96 h (** p < 0.01). Data are presented as the mean ± SD from three independent experiments (n=3).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Flow Cytometry, Staining

AVN944 modulates expression of cell cycle regulators and apoptosis-associated proteins in TC71 cells. (A) Western blot analysis of p53, Cyclin D1, Cyclin E, Bax, Bcl-2, and PARP1 (full-length and cleaved forms) expression in TC71 cells treated with 5 μM AVN944 for 0, 24, and 48 h. α/β-Tubulin was used as a loading control. AVN944 treatment led to a time-dependent increase in expression of p53, Bax, and cleaved PARP1, while there was a marked reduction in expression of Cyclin D1, Cyclin E, Bcl-2, and full-length PARP1. (B-H) Quantification of protein levels, normalized to α/β-Tubulin, using ImageJ software. Protein levels were measured using ImageJ software, normalized to α/β-tubulin, and further normalized to the 0-h time point for each condition. The resulting -fold changes relative to 0-h were annotated directly on the graphs to facilitate quantitative interpretation. The graphs depict relative levels of (B) p53, (C) Cyclin D1, (D) Cyclin E, (E) Bax, (F) Bcl-2, (G) full-length PARP1, and (H) cleaved PARP1. Data are presented as the mean ± SD of three independent experiments (n=3). Statistical significance is indicated (* p < 0.05 and ** p < 0.01).

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: AVN944 modulates expression of cell cycle regulators and apoptosis-associated proteins in TC71 cells. (A) Western blot analysis of p53, Cyclin D1, Cyclin E, Bax, Bcl-2, and PARP1 (full-length and cleaved forms) expression in TC71 cells treated with 5 μM AVN944 for 0, 24, and 48 h. α/β-Tubulin was used as a loading control. AVN944 treatment led to a time-dependent increase in expression of p53, Bax, and cleaved PARP1, while there was a marked reduction in expression of Cyclin D1, Cyclin E, Bcl-2, and full-length PARP1. (B-H) Quantification of protein levels, normalized to α/β-Tubulin, using ImageJ software. Protein levels were measured using ImageJ software, normalized to α/β-tubulin, and further normalized to the 0-h time point for each condition. The resulting -fold changes relative to 0-h were annotated directly on the graphs to facilitate quantitative interpretation. The graphs depict relative levels of (B) p53, (C) Cyclin D1, (D) Cyclin E, (E) Bax, (F) Bcl-2, (G) full-length PARP1, and (H) cleaved PARP1. Data are presented as the mean ± SD of three independent experiments (n=3). Statistical significance is indicated (* p < 0.05 and ** p < 0.01).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: Expressing, Western Blot, Control, Software

In vivo anti-tumor activity of AVN944 in a TC71 xenograft model . (A) The body weight of mice treated with AVN944 (50 mg/kg) or control was monitored over 10 days. There was no significant difference in body weight changes between the AVN944-treated and the control groups, suggestive of no major systemic toxicity. Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9). (B) Volume of tumors in TC71 xenograft-bearing mice treated with AVN944 (50 mg/kg, n = 9) compared with that of tumors in mice treated with the control (n = 6). Tumor growth in the AVN944-treated group was inhibited significantly, and tumor volume remained significantly smaller throughout the experiment (** p < 0.01). (C) Representative images of tumors excised from control (n = 6) and AVN944-treated mice (n = 9) at the end of the experiment. Tumors from AVN944-treated mice were markedly smaller than those from control mice, providing visual confirmation of the significant inhibition of tumor growth observed in Figure B. Each tumor was measured and aligned for comparison, demonstrating a clear reduction in tumor mass after AVN944 treatment . (D) AVN944-induced reductions in tumor weight in TC71 xenografts. Tumor weight was measured post-excision; AVN944 treatment led to a significant reduction in tumor mass when compared with the control (** p < 0.01). Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9).

Journal: International Journal of Biological Sciences

Article Title: AVN944 Elicits Apoptotic Responses and Impedes Tumorigenic Potential in Ewing's Sarcoma Cells

doi: 10.7150/ijbs.116651

Figure Lengend Snippet: In vivo anti-tumor activity of AVN944 in a TC71 xenograft model . (A) The body weight of mice treated with AVN944 (50 mg/kg) or control was monitored over 10 days. There was no significant difference in body weight changes between the AVN944-treated and the control groups, suggestive of no major systemic toxicity. Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9). (B) Volume of tumors in TC71 xenograft-bearing mice treated with AVN944 (50 mg/kg, n = 9) compared with that of tumors in mice treated with the control (n = 6). Tumor growth in the AVN944-treated group was inhibited significantly, and tumor volume remained significantly smaller throughout the experiment (** p < 0.01). (C) Representative images of tumors excised from control (n = 6) and AVN944-treated mice (n = 9) at the end of the experiment. Tumors from AVN944-treated mice were markedly smaller than those from control mice, providing visual confirmation of the significant inhibition of tumor growth observed in Figure B. Each tumor was measured and aligned for comparison, demonstrating a clear reduction in tumor mass after AVN944 treatment . (D) AVN944-induced reductions in tumor weight in TC71 xenografts. Tumor weight was measured post-excision; AVN944 treatment led to a significant reduction in tumor mass when compared with the control (** p < 0.01). Data are presented as the mean ± SD (control: n = 6; AVN944: n = 9).

Article Snippet: By contrast, the TC71 cell line was acquired from the Leibniz Institute DSMZ (Germany).

Techniques: In Vivo, Activity Assay, Control, Inhibition, Comparison

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