hos 143b  (ATCC)

Journal: Cancer Medicine

Article Title: Inhibition of Autophagy Reveals ATR Protein Kinase as a Key Mediator of Cisplatin Sensitivity in Osteosarcoma

doi: 10.1002/cam4.71658

Figure Lengend Snippet: Activation of autophagy in chemotherapy‐treated HOS‐143B cells. (A) Dose response curve for cisplatin treatment. (B) Dose response curve for doxorubicin hydrochloride treatment. The HOS‐143B cells were dosed with either cisplatin or doxorubicin hydrochloride in increasing concentrations up to 200 or 20 μM, respectively. (C) Western blotting images of LC3‐I, LC3‐II, p62, and β‐actin protein levels in 5 μM cisplatin‐treated or 0.75 μM doxorubicin hydrochloride‐treated HOS‐143B cells. Untreated HOS‐143B cells served as the control. β‐actin is used as an internal control. Graphical representation of fold changes in protein levels (D) LC3‐II:LC3‐I and (E) p62 protein levels in cisplatin‐treated and doxorubicin hydrochloride‐treated HOS‐143B cells relative to the control. (F) FITC‐LC3 and DAPI immunofluorescence images of HOS‐143B cells treated with cisplatin (10 μM) or doxorubicin hydrochloride (1 μM) for 48 h. Data are expressed as mean ± SD. For all data n = 3 cell culture replicates run in triplicate; * p ≤ 0.05.

Article Snippet: HOS‐143B (ATCC, CRL‐8303, authenticated) cells were cultured in 1× DMEM + GlutaMAX (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific), 1% non‐essential amino acids (ThermoFisher Scientific), 1% sodium pyruvate (ThermoFisher Scientific), and 1% penicillin–streptomycin (ThermoFisher Scientific).

Techniques: Activation Assay, Western Blot, Control, Immunofluorescence, Cell Culture

Journal: Cancer Medicine

Article Title: Inhibition of Autophagy Reveals ATR Protein Kinase as a Key Mediator of Cisplatin Sensitivity in Osteosarcoma

doi: 10.1002/cam4.71658

Figure Lengend Snippet: KO of ATG7 by CRISPR/Cas9 ablates autophagy in HOS‐143B cells. (Ai) Schematic illustration of the binding site of TrueGuide sgRNA on Exon 11 of ATG7 gene. The sgRNA guides the Cas9 endonuclease to the target site for enzymatic double stranded cleavage ( ThermoFisher.com ). (Aii) Gel electrophoresis of PCR product for the cleavage assay shows a 36% reduction of ATG7 expression in the pooled cell population following ATG7 KO. (B) qRT‐PCR relative expression of ATG7 normalized to GAPDH in ATG7 −/− , NTC and WT HOS‐143B cells following single cell clonal isolation for constitutive knockout of ATG7. (C) Western blotting images of ATG7, LC3‐I, LC3‐II, p62, ATG5‐ATG12, ATG5 and β‐actin protein levels in ATG7 −/− , NTC and WT HOS‐143B cells following single cell clonal isolation for constitutive knockout of ATG7 . Graphical representation of fold changes in relative protein levels of (D) ATG7, (E) LC3‐II:LC3‐I, (F) p62, (G) ATG5‐ATG12, (H) ATG5 in ATG7 −/− and NTC compared to the WT HOS‐143B cells. Data are expressed as mean ± SD. For all data n = 3 biological replicates run in triplicate; **** p < 0.0001; *** p < 0.001; ** p < 0.01 (One‐way ANOVA).

Article Snippet: HOS‐143B (ATCC, CRL‐8303, authenticated) cells were cultured in 1× DMEM + GlutaMAX (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific), 1% non‐essential amino acids (ThermoFisher Scientific), 1% sodium pyruvate (ThermoFisher Scientific), and 1% penicillin–streptomycin (ThermoFisher Scientific).

Techniques: CRISPR, Binding Assay, Nucleic Acid Electrophoresis, Cleavage Assay, Expressing, Quantitative RT-PCR, Single Cell, Isolation, Knock-Out, Western Blot

Journal: Cancer Medicine

Article Title: Inhibition of Autophagy Reveals ATR Protein Kinase as a Key Mediator of Cisplatin Sensitivity in Osteosarcoma

doi: 10.1002/cam4.71658

Figure Lengend Snippet: Chemosensitivity and migration of ATG7 knockout HOS‐143B cells. (A) Western blotting images of LC3‐I, LC3‐II, p62 and β‐actin protein levels in ATG7 −/− , NTC and WT HOS‐143B cells treated with 5 μM of cisplatin or 0.75 μM of doxorubicin hydrochloride. Untreated cells served as the control. To assess chemosensitivity, ATG7 −/− , NTC and WT HOS‐143B cells were dosed with DOX and CIS in increasing concentrations up to 200 μM and 20 μM, respectively. (B) The dose response curve for DOX treatment. (C) Graphical representation of IC 50 of DOX. (D) The dose response curve for CIS treatment. (E) Graphical representation of IC 50 of CIS. To assess migration, a scratch assay was carried out and migratory distance was measured after 48 h of incubation. (F) Graphical representation of migratory distance (μm) of ATG7 −/− , NTC and WT HOS‐143B, in DOX or CIS‐treated and untreated conditions. Data are expressed as mean ± SD. For all data n = 3 biological replicates run in triplicate; ** p < 0.01; * p < 0.05 (One‐way ANOVA).

Article Snippet: HOS‐143B (ATCC, CRL‐8303, authenticated) cells were cultured in 1× DMEM + GlutaMAX (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific), 1% non‐essential amino acids (ThermoFisher Scientific), 1% sodium pyruvate (ThermoFisher Scientific), and 1% penicillin–streptomycin (ThermoFisher Scientific).

Techniques: Migration, Knock-Out, Western Blot, Control, Wound Healing Assay, Incubation

Journal: Cancer Medicine

Article Title: Inhibition of Autophagy Reveals ATR Protein Kinase as a Key Mediator of Cisplatin Sensitivity in Osteosarcoma

doi: 10.1002/cam4.71658

Figure Lengend Snippet: Chemosensitivity of HOS‐143B cells following co‐administration with ATMi/ATRi. (A) Image showing the relative levels of phospho‐p53 (S15; boxed) in ATG7 −/− and NTC HOS‐143B cells. (B) Graphical representation of relative phosphorylation of p53 (S15). For the dose response assay, HOS‐143B cells were dosed with CIS or DOX in increasing concentrations up to 200 μM or 10 μM respectively alongside 1 μM of either ATMi/KU‐60019 or ATRi/VE‐821. (C) Dose response curve for DOX with/without 1 μM ATMi/ATRi. (D) Graphical representation of IC 50 of DOX with/without 1 μM ATMi/ATRi. (E) Dose response curve for CIS with/without 1 μM ATMi/ATRi. (F) Graphical representation of IC 50 values of CIS with/without 1 μM ATMi/ATRi. (G) Dose response curve of CIS combined with/without ATRi A–D. (H) Graphical representation of IC 50 values of CIS with/without ATRi A–D. VE‐821/ATRi A, BAY1895344/ATRi B, AZD6738/ATRi C and AZ20/ATRi D. 1 μM of ATRi A, ATRi C and ATRi D and 0.1 μM of ATRi B. Data are expressed as mean ± SD. For all data n = 3 biological replicates run in triplicate; ** p < 0.01; * p < 0.05 (two‐sample t ‐test vs. control).

Article Snippet: HOS‐143B (ATCC, CRL‐8303, authenticated) cells were cultured in 1× DMEM + GlutaMAX (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific), 1% non‐essential amino acids (ThermoFisher Scientific), 1% sodium pyruvate (ThermoFisher Scientific), and 1% penicillin–streptomycin (ThermoFisher Scientific).

Techniques: Phospho-proteomics, Control

Journal: Cancer Medicine

Article Title: Inhibition of Autophagy Reveals ATR Protein Kinase as a Key Mediator of Cisplatin Sensitivity in Osteosarcoma

doi: 10.1002/cam4.71658

Figure Lengend Snippet: Pharmacological inhibition of ATR enhanced apoptosis and blocked p53 phosphorylation and autophagy. (A) Annexin V/PI staining of HOS‐143B cells with/without 5 μM CIS and/or 0.01 μM ATRi, BAY‐1895344. Quadrant guide: Lower left = live cells; Lower right, early apoptosis; Upper left, necrosis; Upper right, late apoptosis. (B) Percentage of the live, early and late apoptotic and necrotic cell population. (C) Statistical analysis of live, early and late apoptotic and necrotic cell population. (D) Western blotting images of total p53, phospho‐p53 and β‐actin protein levels in HOS‐143B cells treated with either 5 μM CIS or 0.01 μM ATRi, or a combination of both. Untreated cells served as the control. Graphical representation of fold changes in protein levels of (E) Total p53 and (F) phospho‐p53 relative to the control. (G) Western blotting images of ATG7, LC3‐I, LC3‐II, and β‐actin protein levels in HOS‐143B cells treated with either 5 μM CIS or 0.01 μM ATRi, or a combination of both. Untreated cells served as the control. Graphical representation of fold changes in protein levels of (H) ATG7 (I) LC3‐II:LC3‐I, and (J) p62 relative to the control. Data are expressed as mean ± SD. For all data n = 3 biological replicates run in triplicate; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05 (flow cytometry: One‐way ANOVA; Western blot: two‐sample t ‐test vs. control).

Article Snippet: HOS‐143B (ATCC, CRL‐8303, authenticated) cells were cultured in 1× DMEM + GlutaMAX (ThermoFisher Scientific) supplemented with 10% FBS (ThermoFisher Scientific), 1% non‐essential amino acids (ThermoFisher Scientific), 1% sodium pyruvate (ThermoFisher Scientific), and 1% penicillin–streptomycin (ThermoFisher Scientific).

Techniques: Inhibition, Phospho-proteomics, Staining, Western Blot, Control, Flow Cytometry

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