human osteosarcoma cell line  (ATCC)

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A) Immunoblot analyses confirming the presence of endogenous FANCD2 and ectopically expressed FLAG-FANCD2. (B) The FANCD2 complex was immunoprecipitated from U2OS cells expressing FLAG-FANCD2. CBB staining is shown. (C) Immunoblot analyses of FANCD2 knockout (FANCD2 KO ) and ectopically expressed FLAG-FANCD2 (wtD2). ( D ) Immunoblot analyses of EGFP-FANCD2 (WT) and mutant EGFP-FANCD2 (KR) in U2OS cells. (E) Immunoblot analyses confirming mCherry-FANCI and EGFP-FANCD2 in U2OS cells.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Western Blot, Immunoprecipitation, Expressing, Staining, Knock-Out, Mutagenesis

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: ( A ) Functional annotation clustering by biological process. The enrichment score represents the geometric mean (in −log 10 scale) of the p -value in the corresponding annotation cluster. In this GO analysis, an enrichment score of 1.41 indicates a statistically significant difference ( p < 0.05). ( B ) Cellular fatty acid levels were analyzed using gas chromatography/mass spectrometry (GC/MS). The relative levels of each fatty acid (saturated and unsaturated) and total fatty acids are shown (mean ± standard error of the mean (SEM) from 4 independent experiments, one-way analysis of variance coupled with Tukey’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001). Blue: U2OS cells, red: FANCD2 KO cells, green: complemented FANCD2 KO cells (FANCD2 KO +WT). Supplementary Table 2 presents the absolute data.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Functional Assay, Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A, B) Images of EGFP-FANCD2 in medium supplemented with 200 µM oleic acid (OA) are shown. (A) Growth of EGFP-FANCD2 from small dots to a ring-like appearance (yellow arrows). Selected frames from Video 1 are shown. (B) Disappearance of ring-like EGFP-FANCD2. Selected frames from Video 2 are shown. (C) Localization of EGFP-FANCD2 in cells cultured with 200 µM of OA for 2 days. LDs were stained with Lipi-Blue. Scale bars, 5 µm. (D, E) Bar graph showing the percentage of nLDs with FANCD2 in the cell (mean ± SEM from more than 3 independent experiments). (F) Localization of endogenous FANCD2 in U2OS cells cultured with 200 µM of OA for 2 days. Scale bar, 5 µm. (G, H) Correlative light–electron microscopy. U2OS cells stably expressing EGFP-FANCD2 were treated with 100 µM of OA for 24 h. (G) Cells were weakly fixed, and fluorescence images were captured first. Some of the nLDs (magenta) were surrounded by FANCD2 (green). Highly magnified images (yellow squares) are shown below. The upper and lower panels correspond to the images in H (ii) and (iv), respectively. Scale bar, 10 µm. (H) (i) Electron microscopy images of the cell shown in (G) . (ii) (iii) (iv) Highly magnified images (red squares) are shown. Scale bars, 5 µm (i), 1 µm (ii), 200 nm (iii), and 500 nm (iv). Asterisk indicates the nucleolar area.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Cell Culture, Staining, Electron Microscopy, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A) Localization of EGFP-FANCD2 in cells cultured with 200 µM of oleic acid (OA) for 2 days. Lipid droplets (LDs) were stained with Lipi-Blue. Scale bar, 5 µm. ( B , C ) Correlative light–electron microscopy. U2OS cells stably expressing EGFP-FANCD2 were treated with 100 µM of OA for 24 h. ( B ) Cells were weakly fixed, and fluorescence images were captured first. Some nuclear LDs (nLDs; magenta) were surrounded by FANCD2 (green). Highly magnified images (yellow square) are shown below. The upper and lower panels correspond with C (ii) and (iii), respectively. Scale bar, 10 µm. ( C ) (i) Electron microscopy images of the cell shown in ( B ). (ii) and (iii) Highly magnified images (red squares) are shown. Scale bars, 10 µm (i), 1 µm (ii), and 200 nm (iii). The asterisk indicates the condensed chromatin area. ( D ) Localization of endogenous FANCD2 and PML in U2OS cells cultured with 200 µM of OA for 2 days. Orange arrows indicate an nLD with FANCD2 and PML. Representative images are shown. Scale bar, 5 µm. (E, F) The relative EGFP-FANCD2 intensity in the FRAP analysis was calculated using the indicated formulas. The red circles and rectangle indicate the bleached areas. The numbers in panels E and F indicate the following: ( E ) 1, FANCD2; 2, whole nucleus; 3, background and ( F ) 1, FANCD2 with LD; 2, whole nucleus; 3, background; 4, FANCD2 without LD.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Cell Culture, Staining, Electron Microscopy, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A) U2OS cells were cultured with the indicated concentration of oleic acid (OA) for 2 days. DNA damage was induced by treating cells with 500 nM mitomycin C (MMC) for 24 h. The fold change in γH2AX levels relative to the control is shown. Lamin B1 was used as a loading control. (B) The conditions for OA and MMC treatment were the same as those described in (A) . Values below the FANCD2 panel depict the L/S ratios between the monoubiquitinated form (L) and unmodified form (S) of FANCD2. α-tubulin was used as a loading control. (C) Immunofluorescence analysis of FANCD2 (green) and γH2AX (magenta). LDs (cyan) were stained with Lipi-Blue. Cells were exposed to MMC (150 nM) overnight or OA (200 µM) for 2 days. Orange arrows indicate an nLD with FANCD2. Representative images are shown. Scale bar, 5 µm.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Cell Culture, Concentration Assay, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A) Live-cell imaging of U2OS cells expressing both EGFP-FANCD2 and mCherry-FANCI. Lipid droplets (LDs) were stained with Lipi-Blue. Representative images are shown. Scale bar, 5 µm. The conditions for oleic acid (OA) and mitomycin C (MMC) treatment were the same as those described in . (B) LD localization of FANCI after FANCD2 depletion. The culture conditions were the same as those described in . Orange arrows indicate FANCD2 and FANCI colocalization in the nLD. Scale bar, 5 µm. (C) Immunoblot analyses confirming the depletion of FANCD2 proteins. Asterisk indicates monoubiquitinated endogenous FANCI. (D) Bar graph showing the percentage of nLDs with FANCI (mean ± SEM from more than 3 independent experiments). siNC, negative control siRNA; siD2, siFANCD2.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Live Cell Imaging, Expressing, Staining, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: ( A, B ) Co-immunoprecipitation assays were performed using U2OS cells stably expressing FLAG-FANCD2. Total cell extracts were prepared from cells treated with 200 µM oleic acid (OA) for 2 days or 1 µM mitomycin C (MMC) overnight and subjected to immunoprecipitation with anti-FLAG beads. ( B ) L and S indicate the monoubiquitinated and unmodified forms of FANCI, respectively.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Immunoprecipitation, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

doi: 10.1101/2025.02.05.636583

Figure Lengend Snippet: (A) Selected frames are shown. The yellow circle and rectangle indicate the photobleach area. The EGFP intensity in each bleached area was calculated and plotted over time (mean ± standard deviation; see ). The highly magnified images in the middle panels (MMC) represent the bleached areas of the FANCD2 foci. The conditions for oleic acid (OA) and mitomycin C (MMC) treatment were the same as those described in . Scale bars, 5 µm. (B) Fluorescence recovery after photobleaching (FRAP) analysis of EGFP-FANCD2 expressed in U2OS cells (±MMC). Blue and orange symbols indicate the fluorescence recovery of FANCD2 nuclear foci (+MMC) and nucleoplasmic FANCD2 under normal culture conditions (-MMC), respectively. (C) FRAP analyses of EGFP-FANCD2 expressed in U2OS cells (+OA). Blue and orange symbols indicate the fluorescence recovery of FANCD2 in nLDs and nucleoplasmic FANCD2, respectively.

Article Snippet: The human osteosarcoma cell line U2OS and its derivatives were cultured at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco’s modified Eagle medium (Shimadzu Diagnostics) supplemented with 10% fetal bovine serum.

Techniques: Standard Deviation, Fluorescence

Journal: International Journal of Nanomedicine

Article Title: Combined Graphene Oxide with 2-Methoxyestradiol for Effective Anticancer Therapy in-vitro Model

doi: 10.2147/IJN.S498947

Figure Lengend Snippet: Comparison of cytotoxicity of tested samples dissolved in ethanol and DMSO against 143B cells expressed as % of cells viability. Data were analyzed with GraphPad Prism Software version 8.0.1 using bidirectional ANOVA with Tukey’s multiple comparison tests; **p < 0.01, ****p < 0.0001 versus control. The results for the ANOVA test were: p < 0.0001, n=3.

Article Snippet: The human osteosarcoma 143B cell line (ATTC-8303) was purchased from Sigma Aldrich (Poland).

Techniques: Comparison, Software, Control

Journal: International Journal of Nanomedicine

Article Title: Combined Graphene Oxide with 2-Methoxyestradiol for Effective Anticancer Therapy in-vitro Model

doi: 10.2147/IJN.S498947

Figure Lengend Snippet: Cytotoxicity of the tested samples against 143B cells line expressed as % of cell viability (( A ) results for GO; ( B ) results for GO-2ME vs 2ME; ( C ) results for RGO; ( D ) results for RGO-2ME vs 2ME). Data were analyzed with GraphPad Prism Software version 8.0.1 using bidirectional ANOVA with Tukey’s multiple comparison tests; **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. The results for the ANOVA test were: p < 0.0001, n=3.

Article Snippet: The human osteosarcoma 143B cell line (ATTC-8303) was purchased from Sigma Aldrich (Poland).

Techniques: Software, Comparison, Control