human osteosarcoma cell line 143b  (ATCC)

Journal: Drug Delivery

Article Title: Liposome-mediated delivery of a ruthenium-based metallodrug to overcome cisplatin resistance in osteosarcoma

doi: 10.1080/10717544.2026.2671485

Figure Lengend Snippet: Antitumor effect of organometallic compounds in osteosarcoma cell lines. Cell viability (WST-1 assay) in 2D cultures measured after the treatment of 143B Par (A), OS833 Par (B), 143B CisPt-R (C), and OS833 CisPt-R (D) with increasing concentrations of free or encapsulated drugs for 72 h. The effect of equivalent amounts of empty nanoparticles in each cell line is shown. IC 50 values for each treatment are indicated. Error bars represent the standard deviation of three independent experiments. Asterisks indicate statistically significant differences between cisplatin and Ru3 (black) or LIP-Ru3 (salmon) (**** p < 0.0001; two-way ANOVA Tukey's test). (E and F) Analysis of DNA damage (formation of −H2AX foci) in 143B CisPt-R cells treated with the IC 75 of CisPt (8.4 µM), Ru3 (0.9 µM), LIP-Ru3 (0.8 µM), or the equivalent amount of empty nanoparticles (LIP-Empty) for 24 hours. Representative images of immunofluorescence staining (scale bar = 25 µm) (E) and quantification of the formation of gH2AX foci in >100 cells (F) are shown. Asterisks indicate statistically significant differences (** p < 0.01; *** p < 0.001; One-way ANOVA Tukey's test). (G) Cell cycle distribution of 143B CisPt-R cells treated with the IC 50 of CisPt (5 µM), Ru3 (0.5 µM), LIP-Ru3 (0.5 µM), or the equivalent amount of empty nanoparticles (LIP-Empty) for 48 hours. The normalized percentage of cells in each cell cycle phase (mean ± standard deviation of three independent experiments) is shown. Asterisks indicate statistically significant differences in G 0 /G 1 phase (blue) between LIP-empty control and Ru3-formulations and in the G 2 /M phase (pink) between LIP-empty and CisPt (**** p < 0.0001; two-way ANOVA Tukey's test).

Article Snippet: Human osteosarcoma cell line 143B (CRL-8303) was obtained from the ATCC repository (Manassas, VA, USA).

Techniques: WST-1 Assay, Standard Deviation, Immunofluorescence, Staining, Control

Journal: Drug Delivery

Article Title: Liposome-mediated delivery of a ruthenium-based metallodrug to overcome cisplatin resistance in osteosarcoma

doi: 10.1080/10717544.2026.2671485

Figure Lengend Snippet: Cytotoxic effect of organometallic compounds in 3D parental spheroids. Spheroid viability (CTG 3D assay) was measured after the treatment of 143B Par (A and B) and OS833 Par (C and D) spheroids with increasing concentrations of free and encapsulated drugs for 96 h. The effect of equivalent amounts of empty nanoparticles is shown. Representative images of 143B Par (A) and OS833 Par (C) spheroids treated with increasing concentrations of each compound are shown (scale bars = 250 µm). Likewise, cell survival curves and IC50 values (µM) for each condition in 143B Par (B) and OS833 Par (D) spheroids are presented as the mean ± the standard deviation of at least eight biological replicates. Asterisks indicate statistically significant differences between cisplatin and Ru3 (black) or LIP-Ru3 (salmon) (**** p < 0.0001; two-way ANOVA Tukey's test).

Article Snippet: Human osteosarcoma cell line 143B (CRL-8303) was obtained from the ATCC repository (Manassas, VA, USA).

Techniques: Standard Deviation

Journal: Drug Delivery

Article Title: Liposome-mediated delivery of a ruthenium-based metallodrug to overcome cisplatin resistance in osteosarcoma

doi: 10.1080/10717544.2026.2671485

Figure Lengend Snippet: Cytotoxic effect of organometallic compounds in 3D cisplatin-resistant spheroids. Spheroid viability (CTG 3D assay) was measured after the treatment of 143B CisPt-R (A and B) and OS833 CisPt-R (C and D) spheroids with increasing concentrations of free and encapsulated drugs for 96 h. The effect of equivalent amounts of empty nanoparticles is shown. Representative images of 143B CisPt-R (A) and OS833 CisPt-R (C) spheroids treated with increasing concentrations of each compound are shown (scale bars = 250 µm). Cell survival curves and IC50 values (µM) for each condition in 143B CisPt-R (B) and OS833 CisPt-R (D) are presented as the mean ± standard deviation of at least eight biological replicates. Asterisks indicate statistically significant differences between cisplatin and Ru3 (black) or LIP-Ru3 (salmon) (* p < 0.05; ** p < 0.01; **** p < 0.0001; two-way ANOVA Tukey's test).

Article Snippet: Human osteosarcoma cell line 143B (CRL-8303) was obtained from the ATCC repository (Manassas, VA, USA).

Techniques: Standard Deviation

Journal: Drug Delivery

Article Title: Liposome-mediated delivery of a ruthenium-based metallodrug to overcome cisplatin resistance in osteosarcoma

doi: 10.1080/10717544.2026.2671485

Figure Lengend Snippet: Effect of free and nano-encapsulated Ru3 in CSC-enriched 3D osteosarcoma cell cultures. 143B Par cells were plated at low density in tumorsphere medium and left to form tumorspheres for 9 days before being treated for 96 h with increasing concentrations of Ru3, LIP-Ru3, and cisplatin. Treatments with the same amount of empty nanoparticles (LIP-Empty) were also included. Representative images (scale bars = 250 μm) (A), quantification of the number of tumorspheres remaining after the treatment (B), and cell viability (WST-1 assay) (C) are presented. Data is presented as the mean ± the standard deviation of independent replicates. In panel B, black asterisks indicate statistically significant differences between controls and the indicated conditions in each treatment. In panel C, asterisks indicate statistically significant differences between cisplatin and Ru3 (black) or LIP-Ru3 (salmon); (** p < 0.01; *** p < 0.001; one-way (panel B) or two-way (panel C) ANOVA Tukey's test).

Article Snippet: Human osteosarcoma cell line 143B (CRL-8303) was obtained from the ATCC repository (Manassas, VA, USA).

Techniques: WST-1 Assay, Standard Deviation

Journal: Drug Delivery

Article Title: Liposome-mediated delivery of a ruthenium-based metallodrug to overcome cisplatin resistance in osteosarcoma

doi: 10.1080/10717544.2026.2671485

Figure Lengend Snippet: In vivo antitumor effect of free and nanoencapsulated metallic compounds. Established 143B CisPt-R xenografts were randomly assigned to six different groups ( n = 5 per group) and treated i.v. with vehicle (PBS, control), empty liposomes (LIP-Empty), free Ru3 (4 mg/kg, Ru3), ruthenium-loaded liposomes (4 mg/kg, LIP-Ru3), RAPTA-C (4 mg/kg), and cisplatin (4 mg/kg) once a week for 3 weeks (cumulative dose of 12 mg/kg). (A) Curves representing the mean relative tumor volume of 143B CisPt-R xenografts during the treatments. Drug efficacy expressed as the percentage of TGI is indicated. (B) Average tumor weight (left panel) and images of the tumors (right panel) at the end of the experiment (day 20 after the start of the treatment). (C) Change in the body weights of mice during the treatments. Error bars represent the SEM and asterisks indicate statistically significant differences between groups (* p < 0.05; **** p < 0.0001, two-way ANOVA Tukey's test).

Article Snippet: Human osteosarcoma cell line 143B (CRL-8303) was obtained from the ATCC repository (Manassas, VA, USA).

Techniques: In Vivo, Control, Liposomes

Journal: bioRxiv

Article Title: In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

doi: 10.64898/2026.02.19.706776

Figure Lengend Snippet: (A) 143B cells (osteosarcoma) show greater proliferation than hFOB cells (osteoblast control cells) in both high (25 mM) and low (5.6 mM) glucose media in three replicate experiments. (B) Mitochondrial membrane potential of 143B cells was significantly decreased when glucose media was changed from 25 mM to 5.6 mM comparing with hFOB cells in three biological replicate experiments.. (C) Mitochondrial membrane potential of the osteosarcoma cells was surprisingly greater in high glucose but lower in low glucose media. (D) Cell viability of 143B cells normalized to hFOB cells in 5.6 mM glucose or 10 mM galactose medium. Only the 143B osteosarcoma cells had significantly reduced viability in galactose media compared to both hFOB cells in galactose and 143B cells in glucose. (E) Cell viability of 6 osteosarcoma cell lines (including 5 primary tumor derived lines: HOS, MG-63, Saos-2, SJSA-1, U2 OS, and one pulmonary metastasis derived line: 15454-307) grown in 5.6 mM glucose or 10 mM galactose medium. Significant reductions of cell viability to variable degrees were seen in galactose conditions for all cell lines except HOS. Three biological replicate experiments per condition. All boxplots show the median of the data, the boxes mark the limits of the second and third quartiles and the whiskers the smallest and largest values no more than 1.5 times +/- the interquartile range. Points outside that range are considered “outlying” and are plotted individually. Differences between the groups were tested with ANOVA with a post-hoc Tukey test while controlling for biological replicates.

Article Snippet: Authenticated human osteosarcoma cell lines were obtained: 143B (ATCC Cat# CRL-8303, RRID:CVCL_2270), HOS (ATCC Cat# CRL-1543, RRID:CVCL_0312), MG-63 (ATCC Cat# CRL-1427, RRID:CVCL_0426), Saos-2 (ATCC Cat# HTB-85, RRID:CVCL_0548), SJSA-1 (ATCC Cat# CRL-2098, RRID:CVCL_1697), U-2 OS (ATCC Cat# HTB-96, RRID:CVCL_0042) and osteoblast cell lines hFOB 1.19 ((ATCC Cat# CRL-3602, RRID:CVCL_3708), hFOB) and NHOst (Lonza Cat #: CC-2538).

Techniques: Control, Membrane, Derivative Assay

Journal: bioRxiv

Article Title: In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

doi: 10.64898/2026.02.19.706776

Figure Lengend Snippet: Differential expression, tested using DESeq2, and pathway analysis, tested using fgsea, is shown for 143B (A, B), HOS (C, D), MG-63 (E, F), Saos-2 (G, H), SJSA1 (I, J), U-2 OS (K, L) and osteosarcoma lung metastasis 15454-307 (M, N) osteosarcoma cell lines. Volcano plots display differential expression results with log2 fold change on the x-axis and negative log10 adjusted p-value on the y-axis (A, C, E, G, I, K, M). Bar plots detail the top five most up- and down-regulated pathways (B, D, F, H, J, L, N). Both significantly different points in the volcano plot and significant pathways in the bar plots are colored by cell line; non-statistically significant points or bars are shown in gray. Legends are grouped, with the legend applying to both the volcano plot and the bar plot of the same color.

Article Snippet: Authenticated human osteosarcoma cell lines were obtained: 143B (ATCC Cat# CRL-8303, RRID:CVCL_2270), HOS (ATCC Cat# CRL-1543, RRID:CVCL_0312), MG-63 (ATCC Cat# CRL-1427, RRID:CVCL_0426), Saos-2 (ATCC Cat# HTB-85, RRID:CVCL_0548), SJSA-1 (ATCC Cat# CRL-2098, RRID:CVCL_1697), U-2 OS (ATCC Cat# HTB-96, RRID:CVCL_0042) and osteoblast cell lines hFOB 1.19 ((ATCC Cat# CRL-3602, RRID:CVCL_3708), hFOB) and NHOst (Lonza Cat #: CC-2538).

Techniques: Quantitative Proteomics

Journal: bioRxiv

Article Title: In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

doi: 10.64898/2026.02.19.706776

Figure Lengend Snippet: Cell viability of (A) healthy osteoblasts hFOB, (B) primary osteosarcomas 143B and (C) MG-63, and (D) lung metastasis 15454-307 treated with 5 µM cycloheximide. Cell viability of (E) healthy osteoblasts NHOst, (F) healthy fibroblasts Q2148, and osteosarcomas (G) MG-63 or (H) 15454-307 treated with metformin, ONC201, or ONC206 in 5.6 mM glucose DMEM-10% FBS medium for 72 h. Cell viability of the same control cells and osteosarcoma cells, (I) hFOB, (J) NHOst, (K) Q2148, (L) 143B, (M) MG-63, (N) 15454-307 treated with 1.5 µM ONC201, or 0.15 µM ONC206 in 17 mM glucose DMEM-10%FBS medium for 72 h. Cell viability was tested using ANOVA with a post-hoc Tukey test for all comparisons. Box plots identify the median second and third quartiles and the whiskers extend to the smallest and largest values no more than 1.5 times +/- the interquartile range. Points outside that range are considered “outlying” and are plotted individually.

Article Snippet: Authenticated human osteosarcoma cell lines were obtained: 143B (ATCC Cat# CRL-8303, RRID:CVCL_2270), HOS (ATCC Cat# CRL-1543, RRID:CVCL_0312), MG-63 (ATCC Cat# CRL-1427, RRID:CVCL_0426), Saos-2 (ATCC Cat# HTB-85, RRID:CVCL_0548), SJSA-1 (ATCC Cat# CRL-2098, RRID:CVCL_1697), U-2 OS (ATCC Cat# HTB-96, RRID:CVCL_0042) and osteoblast cell lines hFOB 1.19 ((ATCC Cat# CRL-3602, RRID:CVCL_3708), hFOB) and NHOst (Lonza Cat #: CC-2538).

Techniques: Control

Journal: bioRxiv

Article Title: In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

doi: 10.64898/2026.02.19.706776

Figure Lengend Snippet: 2500 cells per well were seeded in 96 well plates with DMEM 10% FBS 12.5 mM glucose medium, and, after culturing overnight, the media was removed and replaced with the same medium with or without 25 nM antimycin A (AA) alone or with 2 mM dichloroacetate (DCA) alone or with a combination of the same concentrations of AA and DCA in (A) healthy osteoblast hFOB cells, (B) primary osteosarcoma 143B or (C) MG63 cells. (D-G) Cells were treated with a combination of 2.5 µM ONC201 (201) and 0.25 µM ONC206 (206), either with just the two-drug treatment or with the addition of 2 mM DCA, or 2 mM metformin (met) in 200 µL medium per well in (D) hFOB, (E) 143B, (F) MG-63 or (G) 15454-307 cells. (H-K) Cells were also treated with a combination of 2.5 µM ONC201, 2 mM DCA, and 2 mM met in 200 µL medium per well in (H) hFOB, (I) 143B, (J) MG-63 or (K) 15454-307 cells. After cells were cultured for 6 days, then MTT cell viability assays were performed with n = 3 biological replicate experiments. Cell viability was tested using ANOVA with a post-hoc Tukey test for all comparisons. Box plots identify the median second and third quartiles and the whiskers extend to the smallest and largest values no more than 1.5 times +/- the interquartile range. Points outside that range are considered “outlying” and are plotted individually.

Article Snippet: Authenticated human osteosarcoma cell lines were obtained: 143B (ATCC Cat# CRL-8303, RRID:CVCL_2270), HOS (ATCC Cat# CRL-1543, RRID:CVCL_0312), MG-63 (ATCC Cat# CRL-1427, RRID:CVCL_0426), Saos-2 (ATCC Cat# HTB-85, RRID:CVCL_0548), SJSA-1 (ATCC Cat# CRL-2098, RRID:CVCL_1697), U-2 OS (ATCC Cat# HTB-96, RRID:CVCL_0042) and osteoblast cell lines hFOB 1.19 ((ATCC Cat# CRL-3602, RRID:CVCL_3708), hFOB) and NHOst (Lonza Cat #: CC-2538).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: In vitro modeling of nutritional and mitochondria-targeted therapies for osteosarcoma

doi: 10.64898/2026.02.19.706776

Figure Lengend Snippet: (A-B) Looking at pediatric cancers with Clp alterations, (A) 1.15% of osteosarcomas have ClpP alterations and (B) 9.20% of osteosarcomas have ClpX alterations. Cancers are given on the x-axis with the y-axis the percentage of alterations across all patients in either (A) ClpP or (B) ClpX. Color indicates the alteration type. (C-E) 90% confluent hFOB or 143B cells in 100 mm Petri dishes were treated with increasing doses of ONC201 or ONC206 indicated in 5.6 mM glucose DMEM, after 24 h, cells were collected and lysed in RIPA buffer. (C) Western immunoblots following SDS-PAGE (immunoblots for ClpP and ClpX are in supplemental figure S4) were imaged with anti-ClpP o r ClpX and anti-CS antibodies, resulting band intensities for the untreated and highest dose of each drug were analyzed by ImageJ, normalized to CS and relative to hFOB for ClpP (D) and ClpX (E) . (F) The differential expression results from 143B cells treated with ONC201 or ONC206 were used to evaluate the overlap in expression changes using an UpSet plot, where the bars show the number of genes changed and a single dot underneath for ONC201 and ONC206, respectively, and the line showing the number of significantly differentially expressed genes in common with both treatments. Pathway analysis, done with over-representation analysis (ORA) in WebGestaltR, on gene expression changes unique to (G) ONC201 alone, (H) ONC206 alone, (I) or common to both versions of the drug. Pathway analysis bar plots give the top five most up- and down-regulated pathways colored by treatment type with non-statistically significant pathways in gray (G-I) .

Article Snippet: Authenticated human osteosarcoma cell lines were obtained: 143B (ATCC Cat# CRL-8303, RRID:CVCL_2270), HOS (ATCC Cat# CRL-1543, RRID:CVCL_0312), MG-63 (ATCC Cat# CRL-1427, RRID:CVCL_0426), Saos-2 (ATCC Cat# HTB-85, RRID:CVCL_0548), SJSA-1 (ATCC Cat# CRL-2098, RRID:CVCL_1697), U-2 OS (ATCC Cat# HTB-96, RRID:CVCL_0042) and osteoblast cell lines hFOB 1.19 ((ATCC Cat# CRL-3602, RRID:CVCL_3708), hFOB) and NHOst (Lonza Cat #: CC-2538).

Techniques: Western Blot, SDS Page, Quantitative Proteomics, Expressing, Gene Expression

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a The relative copy number, mRNA expression of ASB7 and cell line count of each tumor type were plotted using 25Q2 DepMap cancer cell line data. b RNA-seq analysis of ASB7 mRNA levels in normal ( n = 4) and osteosarcoma ( n = 16) tissues. c Kaplan-Meier survival curves comparing osteosarcoma patients with low versus high ASB7 protein expression (low, n = 19; high, n = 19). The cutoff value for distinguishing the low- and high-expression groups was defined as the median IHC score obtained from the HALO system. The corresponding P value from the log-rank test is shown. d , e , h 143B cells stably expressing doxycycline-inducible ASB7 overexpression were treated with or without 0.5 μg/mL doxycycline for 24 h. After that, the cells were subjected to western blotting to verify the upregulation of ASB7 expression ( d ), immunofluorescence staining with Actin-Tracker ( e ), and migration and invasion assays ( n = 3 independent experiments) ( h ). f The percentage of cells with protrusions was quantified in 143B cells overexpressing ASB7. Cells exhibiting finger-like filopodia (marked by asterisks) or fan-/wave-like lamellipodia (indicated by arrowheads), as shown in e , were scored as positive. g GO analysis of genes upregulated in 143B cells overexpressing ASB7. i – l Luciferase-expressing 143B cells with a doxycycline-inducible ASB7 overexpression system were injected into mouse tibiae. After that, the mice were fed either a control diet or a diet containing 0.065% doxycycline. Lung metastases were assessed by IVIS imaging ( i ) and quantitative bioluminescence analysis ( j ). Lung tissue sections were subjected to H&E staining ( k ), and metastatic foci were quantified ( l ). n = 5 mice per group. m – q 143B cells expressing sgRNAs of either non-target control or ASB7 were subjected to western blotting to detect the expression of ASB7 ( m ), immunofluorescence staining with Actin-Tracker ( n ), a quantification of cells with finger-like or fan-/wave-like protrusions ( o ), migratory and invasive assays ( n = 3 independent experiments) ( p ), and an MTT assay ( n = 3 independent experiments) ( q ). r – u Mice implanted with 143B cells expressing both luciferase and sgRNAs targeting either non-target control or ASB7 were analyzed by bioluminescence imaging, IVIS images are shown ( r ), and the data are plotted ( s ). Lung sections from these mice were analyzed by H&E staining ( t ), and the quantification of metastatic foci ( u ) is plotted. n = 5 mice per group. Data are shown as the mean ± S.D. P values were derived from unpaired two-tailed Student’s t -test for the data in b , f , h , j , l , o , p , s , u and from two-way ANOVA followed by Tukey’s multiple comparisons test for the data in q .

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Expressing, RNA Sequencing, Stable Transfection, Over Expression, Western Blot, Immunofluorescence, Staining, Migration, Luciferase, Injection, Control, Imaging, MTT Assay, Derivative Assay, Two Tailed Test

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a The normalized protein levels of ASB7 and ATF2 across unbiased re-classified tumor subtypes were plotted using data from the UALCAN database , . Pearson’s correlation coefficient ( r ) was calculated to assess the relationship between ASB7 and ATF2 protein levels across tumor subtypes. b Representative images of immunohistochemical staining of ASB7 and ATF2 in osteosarcoma tissues. c A contingency table was constructed to display the stratification of 38 osteosarcoma cases based on high or low expression of ASB7 and ATF2, in which the dichotomization was determined by the median IHC score obtained from the HALO system. The Spearman correlation coefficient was used to quantify the association, while Pearson’s chi-square test was used to determine the corresponding χ 2 statistic and P value. d Kaplan-Meier survival analysis of osteosarcoma patients based on ATF2 protein expression level (low, n = 19; high, n = 19). The P value from the log-rank test is shown, with the high and low-expression groups dichotomized by the median IHC score obtained from the HALO system. e – j 143B cells expressing vector or ATF2 were subjected to western blotting ( e ), Actin-Tracker staining ( f ), quantification of cells exhibiting finger-like or fan-/wave-like protrusions ( g ), cell migration and invasion assays ( n = 3 independent experiments) ( i ), and MTT assays ( n = 3 independent experiments) ( j ). h GO analysis of genes upregulated in 143B cells in which ATF2 was knocked down by siRNAs. k , l In vivo fluorescence imaging and the corresponding quantitative analysis ( k ), H&E staining and the corresponding quantitative analysis ( l ) of mice bearing orthotopic 143B cells expressing luciferase together with either vector or ATF2 ( n = 5 mice per group). m – q In 143B cells expressing sgRNAs targeting either non-target control or ATF2, western blotting ( m ), Actin-Tracker staining ( n ), quantification of cells with finger-like or fan-/wave-like protrusions ( o ), cell migration and invasion assays ( n = 3 independent experiments) ( p ), and MTT assays ( n = 3 independent experiments) ( q ) were performed. r, s In mice bearing orthotopic 143B cells expressing luciferase together with sgRNAs targeting non-target control or ATF2, in vivo fluorescence imaging ( r ), H&E staining ( s ) of lung sections and the corresponding quantitative analyses were performed ( n = 5 mice per group). Data are shown as the mean ± S.D. P values were derived from unpaired two-tailed Student’s t -test for the data in g , i , k , l , o , p , r , s ; from two-way ANOVA followed by Sidak’s test for the data in j ; or from two-way ANOVA followed by Tukey’s test for the data in q .

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Immunohistochemical staining, Staining, Construct, Expressing, Plasmid Preparation, Western Blot, Migration, In Vivo, Fluorescence, Imaging, Luciferase, Control, Derivative Assay, Two Tailed Test

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a , b ATF2 protein levels were analyzed by western blotting in 143B cells with ASB7 overexpression ( a ) or knockout ( b ). c Western blotting analysis of ATF2 protein levels in 143B cells treated with MG132 (10 μM, 6 h), MLN4924 (1 μM, 12 h), or bafilomycin A1 (200 nM, 6 h). d – g Western blotting analysis of ATF2 protein levels at the indicated times following CHX (40 μg/mL) treatment in 143B cells with ASB7 overexpression ( d ) or knockout ( f ). Panels e and g show the corresponding quantitative data from these experiments. n = 3 independent experiments. h HEK293T cells were co-transfected with the indicated constructs for 24 h and treated with 10 μM MG132 for another 6 h. After that, the cells were lysed and subjected to immunoprecipitation using anti-Flag or anti-V5 beads. i HEK293T cells were co-transfected with the indicated constructs for 24 h and subsequently treated with MG132 (10 μM, 6 h). After that, the cells were subjected to a Ni-NTA pull-down assay to examine the exogenous ubiquitination of ATF2. j , k Endogenous ubiquitination of ATF2 was evaluated by Ni-NTA pull-down assay in 143B cells with ASB7 overexpression ( j ) or knockout ( k ) following transfection with His-Ub and treatment with 10 μM MG132 for 6 h. l Ubiquitination analysis of ATF2 mutants. HEK293T cells were co-transfected with the indicated constructs, treated with MG132 (10 μM, 6 h), and subjected to a Ni-NTA pull-down assay to assess ATF2 ubiquitination levels. Data are shown as the mean ± S.D. P values were derived from two-way ANOVA followed by Sidak’s multiple comparisons test, as shown in ( e ), or two-way ANOVA followed by Tukey’s multiple comparisons test, as shown in g .

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Western Blot, Over Expression, Knock-Out, Transfection, Construct, Immunoprecipitation, Pull Down Assay, Ubiquitin Proteomics, Derivative Assay

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a GO analysis of genes upregulated in both 143B cells with ASB7 overexpression and those with ATF2 knockdown by siRNAs. b – e 143B cells expressing doxycycline-induced ASB7 together with either vector or ATF2 were subjected to western blotting ( b ), Actin-Tracker staining ( c ), quantification of cells with finger-like or fan-/wave-like protrusions ( d ), and migration and invasion assays ( e ). n = 3 independent experiments. f – i In mice bearing orthotopic 143B-Luc cells expressing doxycycline-induced ASB7 together with either vector or ATF2, in vivo lung fluorescence images ( f ) and H&E staining of lung sections ( h ) are shown, along with the corresponding quantitative analyses in g , i , respectively. n = 5 mice per group. Data are shown as the mean ± S.D. Statistical significance in d , e , g , i was assessed by an unpaired two-tailed Student’s t -test.

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Over Expression, Knockdown, Expressing, Plasmid Preparation, Western Blot, Staining, Migration, In Vivo, Fluorescence, Two Tailed Test

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a The ATF2 peak enrichment scores and expression fold changes for migration-related genes upregulated upon ASB7 overexpression were plotted. The dashed red line indicates the ideal distribution of genes that are both strongly bound by ATF2 and upregulated upon ASB7 overexpression. b Pearson’s correlation coefficients ( r ) and the corresponding P values of ATF2 and ITGB2 expression across tumor types were plotted using TCGA transcriptomic data from the GEPIA database. c Schematic of the ITGB2 promoter region (top) and the subsequent ChIP-qPCR assay used to identify the site enriched in ATF2 in 143B cells. d – k ITGB2 protein levels were analyzed by western blotting in 143B cells with ASB7 overexpression ( d ), ASB7 knockout ( f ), ATF2 overexpression ( h ), and ATF2 knockout ( j ), respectively. ITGB2 mRNA levels were analyzed by qRT-PCR in 143B cells with ASB7 overexpression ( e ), ASB7 knockout ( g ), ATF2 overexpression ( i ), and ATF2 knockout ( k ), respectively. l – p 143B cells expressing vector or ITGB2 were analyzed using western blotting ( l ) and Actin-Tracker staining ( m ). The proportion of cells with finger-like or fan-/wave-like protrusion morphology was quantified ( n ). Additionally, cell migration and invasion assays ( o ) and MTT assays ( p ) were performed. q – u 143B cells expressing sgRNAs targeting either non-target control or ITGB2 were subjected to western blotting ( q ), Actin-Tracker staining ( r ), quantification of cells with finger-like or fan-/wave-like protrusions ( s ), cell migration and invasion assays ( t ), and MTT assays ( u ). The data are shown as the mean ± S.D. from three independent experiments. P values were derived from unpaired two-tailed Student’s t -test for the data in c , e , g , i , k , n , o , s , t and from two-way ANOVA followed by either Sidak’s or Tukey’s multiple comparisons tests for the data in p , u , respectively.

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Expressing, Migration, Over Expression, ChIP-qPCR, Western Blot, Knock-Out, Quantitative RT-PCR, Plasmid Preparation, Staining, Control, Derivative Assay, Two Tailed Test

Journal: Cell Discovery

Article Title: ASB7 promotes osteosarcoma lung metastasis through ubiquitin-mediated degradation of ATF2

doi: 10.1038/s41421-026-00890-9

Figure Lengend Snippet: a HEK293T cells were co-transfected with V5-ATF2 and Flag-HDACs, and the interaction between ATF2 and HDACs was assessed by immunoprecipitation with anti-Flag beads. b qRT-PCR analysis of ITGB2 mRNA levels in 143B cells after knockdown of HDAC3, HDAC6, or HDAC10. c qRT-PCR analysis of ITGB2 mRNA levels in 143B cells with the indicated constructs. d Co-immunoprecipitation analysis of the endogenous interaction between HDAC6 and ATF2 in 143B cells. e , f 143B cells stably expressing vector or HDAC6 were analyzed by western blotting ( e ) and cell migration and invasion assays ( n = 3 independent experiments) ( f ). g , h 143B cells expressing shRNAs targeting either non-target control or HDAC6 were subjected to western blotting ( g ) and cell migration and invasion assays ( n = 3 independent experiments) ( h ). i Heatmaps showing the genome-wide distributions of ATF2 ChIP-seq signals (ENCODE) and HDAC6 CUT&Tag signals across gene bodies with ± 3.0 kb flanking regions. j Heatmaps showing the distributions of ATF2 ChIP-seq signals (ENCODE) and HDAC6 CUT&Tag signals centered on the TSSs of ATF2-bound promoters associated with migration-related genes. The signals are plotted ± 3.5 kb around the TSS. k , l ChIP-qPCR analysis of HDAC6 enrichment at the ITGB2 promoter in 143B cells with ATF2 overexpression ( k ) or knockout ( l ). m Model of the ASB7-ATF2/HDAC6-ITGB2 axis in osteosarcoma. ATF2 recruits HDAC6 to transcriptionally repress ITGB2 and inhibit cell movement, while ASB7-mediated ATF2 degradation relieves this repression, activating ITGB2 expression and inducing protrusion formation to drive metastasis. The data are shown as the mean ± S.D. from three independent experiments. P values were derived from unpaired two-tailed Student’s t -test, as shown in b , c , f , h , k , l .

Article Snippet: The human cell lines 143B and HEK293T were purchased from ATCC.

Techniques: Transfection, Immunoprecipitation, Quantitative RT-PCR, Knockdown, Construct, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Migration, Control, Genome Wide, ChIP-sequencing, ChIP-qPCR, Over Expression, Knock-Out, Derivative Assay, Two Tailed Test