u-2os (human osteosarcoma cell line)  (Thermo Fisher)

Journal: Cellular & Molecular Biology Letters

Article Title: High-content image screening to identify chemical modulators for peroxisome and ferroptosis

doi: 10.1186/s11658-024-00544-2

Figure Lengend Snippet: High-content image analysis of cell-cycle-dependent peroxisomal signal in U-2OS cells expressing the mOrange2-Peroxisomes2 marker. A Representative image of U-2OS cell line with mOrange2-Peroxisomes2 fluorescent label, scale bar: 100 μm. B High-content image analysis workflow of single-cell detection and peroxisomal signal quantification. Single cell nuclei were detected based on the Hoechst staining (blue markers), while clustered nuclei or the nuclei at the edge of images (yellow markers) were excluded from analysis (primary object identification). Afterward, selected single cells were segmented into cytoplasmic and nuclear regions (nuc/cyto segmentation), and the peroxisomal signal in the cytoplasm was quantified (peroxi spot identification). C The peroxisomal signals after the treatment with compounds (1 μM, 24 h) from the target-selective chemical library were normalized to the control average (DMSO treatment). Blue: compounds reducing peroxisomal signals. Red: compounds increasing peroxisomal signals. D Histogram showing the distribution of Hoechst DNA staining signals in the control cell population. E Peroxisomal spot total intensity was plotted against the Hoechst signal in individual cells of the control sample. The distribution of peroxisomal spot total intensity per cell was summarized in cell populations of different cell cycle phases. F Flow cytometry analysis of U-2OS cells with peroxisomal fluorescent marker and Hoechst staining. The relative levels of peroxisomal intensities in each cell cycle phases were normalized against G1 cell population. G U-2OS cells were treated with aphidicolin (DNA synthesis inhibitor, 1 μM), VX-680 (Aurora Kinase inhibitor, 1 μM) or aphidicolin plus VX-680 for 24 h. The cell cycle distribution and peroxisomal signals were analyzed by flow cytometry. The relative levels of peroxisomal intensities in each sample were normalized against the control cell population. n = 3 independent experiments. * p < 0.05; *** p < 0.001

Article Snippet: Human osteosarcoma cell line U-2OS (catalog no. CL-0236) and human cervical cancer cell line HeLa (catalog no. CL-0101) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Marker, Staining, Flow Cytometry, DNA Synthesis

Journal: Cellular & Molecular Biology Letters

Article Title: High-content image screening to identify chemical modulators for peroxisome and ferroptosis

doi: 10.1186/s11658-024-00544-2

Figure Lengend Snippet: High-content screening results of a target-selective library in U-2OS cells expressing the mOrange2-Peroxisomes2 marker. A The average peroxisomal signal was plotted against Hoechst staining intensity for each chemical treatment. The signal of each channel was standardized by the mean value of the control sample. Data are summarized from three biological replicates. Group A: compounds that affect peroxisomes by altering cell cycle. Group B: compounds that increase peroxisomal abundance without altering cell cycle. Group C: compounds that reduce peroxisomal abundance without altering cell cycle. B Representative results of Hoechst intensity distribution and the relative peroxisomal signals in control and compounds from each group. C Heatmap showing the relative values (normalized to controls) of cell counts, peroxisomal spot counts per cell, peroxisomal total intensity per cell, and Hoechst intensity per cell of compound treatment in group A–C. D Representative images of control and chemical treatment from each group: Panobinostat: group A; CEP-18770 and KPT-330: group B; Naftopidi and AZD6738: group C. Scale bar: 100 μm

Article Snippet: Human osteosarcoma cell line U-2OS (catalog no. CL-0236) and human cervical cancer cell line HeLa (catalog no. CL-0101) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: High Content Screening, Expressing, Marker, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: High-content image screening to identify chemical modulators for peroxisome and ferroptosis

doi: 10.1186/s11658-024-00544-2

Figure Lengend Snippet: Validation of screening results by immunofluorescence (IF) staining and Western blot. A IF staining of intrinsic peroxisomal marker PMP70 in HeLa and U-2OS cell lines. High-content imaging analysis was applied to quantify PMP70 staining intensity after the treatment of selective compounds (1 μM, 24 h). Dotted line represents the mean value of control samples (DMSO treatment). Data are summarized from three biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.001. B Western blot analysis of PEX3 protein levels after the treatment of indicated compounds (1 μM, 24 h) in HeLa and U-2OS cell lines

Article Snippet: Human osteosarcoma cell line U-2OS (catalog no. CL-0236) and human cervical cancer cell line HeLa (catalog no. CL-0101) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Marker, Imaging

Journal: Cellular & Molecular Biology Letters

Article Title: High-content image screening to identify chemical modulators for peroxisome and ferroptosis

doi: 10.1186/s11658-024-00544-2

Figure Lengend Snippet: Peroxisomal abundance and oxidative stress. A Flow cytometry analysis of DCFDA staining (ROS sensor) in U-2OS cells after treatment with compounds that promoted peroxisomal abundance. B Flow cytometry analysis of DCFDA staining (ROS sensor) in U-2OS cells after treatment with compounds that reduced peroxisomal abundance. C U-2OS cells with peroxisomal fluorescent tag were treated with 1 μM panobinostat (HDAC inhibitor), pacritinib (JAK inhibitor), and NSC697923 (E2 conjugating enzyme inhibitor), MLN2238 (proteasome inhibitor) and KPT-185 (CRM1 inhibitor) in presence or absence of 2 mM NAC. The relative levels of peroxisomal signals were analyzed by flow cytometry. N = 3 independent experiments. * p < 0.05 versus control; ** p < 0.01 versus control; *** p < 0.001 versus control; # p < 0.05 versus treatment; ## p < 0.01 versus treatment

Article Snippet: Human osteosarcoma cell line U-2OS (catalog no. CL-0236) and human cervical cancer cell line HeLa (catalog no. CL-0101) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Flow Cytometry, Staining

Journal: Cellular & Molecular Biology Letters

Article Title: High-content image screening to identify chemical modulators for peroxisome and ferroptosis

doi: 10.1186/s11658-024-00544-2

Figure Lengend Snippet: Peroxisome-promoting compounds sensitize U-2OS cells to ferroptosis induction. A U-2OS cells were treated with ferroptosis inducer (RSL3 (1 μM) or erastin (2 μM)), proteasome inhibitor MLN2238 (1 μM) or drug combination with/without ferroptosis inhibitor Fer-1 (2 μM) for 24 h. Cell death events were analyzed by PI staining. B Summary of percentage of cell death events in U-2OS cells after treatment with ferroptosis inducer (1 μM RSL3 or 2 μM erastin), chemicals (1 μM) or drug combination with/without ferroptosis inhibitor Fer-1 (2 μM). N = 3 independent experiments. *** p < 0.001 versus the Fer-1 treatment group; ## p < 0.01 and ### p < 0.001 versus RSL3 treatment; $$ p < 0.01 and $$$ p < 0.001 versus erastin treatment; &&& p < 0.001 versus control group. C U-2OS cells with or without MLN2238 treatment (1 μM, 24 h) were exposed to 200 μM H 2 O 2 for 30 min. Cells were labeled with DCFDA, and the relative ROS levels were determined by flow cytometry. N = 3 independent experiments. * p < 0.05; ** p < 0.01. D Spheroids derived from U-2OS cells were treated with RSL3 (1 μM), MLN2238 (1 μM) or RSL3 + MLN2238 for 24 h. The spheroids were then stained with PI and imaged under EVOS 7000 microscope, scale bar: 100 μm

Article Snippet: Human osteosarcoma cell line U-2OS (catalog no. CL-0236) and human cervical cancer cell line HeLa (catalog no. CL-0101) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Staining, Labeling, Flow Cytometry, Derivative Assay, Microscopy