Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and C ) Representative microscopic images of BrdU incorporation assays in U2OS and MG63 osteosarcoma cells under normoxic (control) and CoCl 2 induced hypoxic conditions. Scale bar = 50 μm. ( B and D ) Quantification of BrdU-positive cells showing a significant decrease in proliferation in hypoxic U2OS and MG63 cells compared with their respective controls (n ≥ 20 cells per condition). ( E and G ) Representative images of colony-formation assays in control and CoCl₂-treated hypoxic U2OS and MG63 cells, respectively. (F and H) Quantification of colony numbers showing a marked reduction in the clonogenic potential of hypoxic osteosarcoma cells. ( I and K ). Representative images of cell-migration assays in U2OS and MG63 cells under control and hypoxic conditions. Scale bar = 50 µm. ( J and L) Quantification of migrated cells showed a significant reduction in the migratory capacity of hypoxic U2OS and MG63 cells, respectively. Statistical significance was calculated using two-tailed Student’s t -test and is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: BrdU Incorporation Assay, Control, Migration, Two Tailed Test

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) U2OS cells, either stably expressing K294A upon doxycycline induction or uninduced controls, were biochemically fractionated into nuclear and cytoplasmic compartments. Western blot analysis confirmed fractionation quality using Lamin A/C as a nuclear marker and GAPDH as a cytoplasmic marker. Myc blotting verified the expression of K294A upon doxycycline induction. ( B ) Quantitative real-time PCR analysis revealed a significant reduction in the cytoplasmic levels of RORA and KCTD16 transcripts in K294A expressing cells compared with controls, while BNIP3 levels remained unchanged. ( C ) Western blot analysis further confirmed decreased protein levels of RORA and KCTD16 in K294A-expressing cells. Myc blotting verified stable K294A expression. ( D ) Densitometric analysis of RORA and KCTD16 protein bands from panel C was performed using ImageJ software. β-Actin was used as a loading control for normalization. Statistical analysis was calculated by performing two-tailed Student’s t -test. ( E ) Table summarizing the internal CAGE (Cap Analysis of Gene Expression) sites identified within the analysed transcripts, with the specific positions highlighted in red. ( F - J ) Bar graphs representing the genomic distribution of CAGE peaks for each gene, illustrating the relative frequency of CAGE signals across different transcript regions. ( K ) Schematic illustration of the Xrn1 susceptibility assay used to assess the stability of 5′-capped transcripts. ( L ) Relative 5′-end loss of RORA and KCTD16 was assessed using an in vitro Xrn1 susceptibility assay. In K294A-expressing cells, both transcripts exhibited a level of 5′-end loss comparable to STAT3 , a known cCE target, relative to control cells. Statistical analysis was performed using one sample Student’s t -test. ( M ) Western blot analysis showing Xrn1 protein levels in Xrn1 knockdown cells with or without doxycycline-induced K294A expression. Myc detection confirmed successful induction of the dominant-negative cCE mutant. ( N ) Quantification of Xrn1 knockdown efficiency was performed using ImageJ software, with β-Actin serving as the internal loading control. ( O ) RORA and KCTD16 exhibited the most pronounced rescue in Xrn1 knockdown cells expressing K294A, indicating their strong dependence on cytoplasmic capping for stability. Statistical significance was determined using one-way ANOVA. All the data is represented as mean ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Stable Transfection, Expressing, Western Blot, Fractionation, Marker, Real-time Polymerase Chain Reaction, Software, Control, Two Tailed Test, Gene Expression, Drug Susceptibility Assay, In Vitro, Knockdown, Dominant Negative Mutation, Mutagenesis

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and B ) qPCR analysis of the selected transcripts in U2OS and MG63 cells revealed reduced expression following treatment with the HIF1α inhibitor PX478, irrespective of hypoxia induction. ( C and E ) Western blot analysis of U2OS and MG63 cells demonstrated reduced protein levels of all selected targets, including HIF1α, in PX478-treated hypoxic samples. ( D and F ) Quantification of western blot band intensities corresponding to panels C and E was performed using ImageJ software. β-Actin served as the loading control for normalization. Data are presented as mean ± SD from three biological replicates. Statistical significance was determined using one-way ANOVA. ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Expressing, Western Blot, Software, Control

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A ) Western blot analysis of c-Myc protein levels in U2OS cells under normoxic and CoCl₂-induced hypoxic conditions. ( B ) Densitometric quantification of c-Myc expression from panel A using ImageJ, showing reduced c-Myc levels in hypoxic U2OS cells. β-Actin served as the loading control. Statistical significance was determined using two-tailed Student’s t -test. ( C and E ) Western blots showing siRNA-mediated depletion of RORA ( C ) and KCTD16 ( I ) in U2OS and MG63 cells under hypoxic conditions. HIF1α blot confirms hypoxia induction. c-Myc levels were elevated upon depletion of either gene. ( D and J ) ImageJ-based densitometric quantification of blots from panels C and I , normalized to β-actin. Statistical analysis was performed using one-way ANOVA. ( E and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 depleted hypoxic U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( F and L ) Quantification of BrdU-positive cells showing increased proliferation upon RORA or KCTD16 depletion under hypoxic conditions (n ≥ 20 cells per condition). ( G and M ) Representative images of colony formation assays in RORA-and KCTD16-depleted hypoxic osteosarcoma cells. ( H and N ) Quantitative analysis showing enhanced clonogenic potential following RORA or KCTD16 depletion in hypoxic cells. Statistical significance was calculated using one-way ANOVA. All the data is represented as ± SD from three biological replicates. ns: non-significant, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Expressing, Control, Two Tailed Test, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Cytoplasmic capping enzyme targeted, hypoxia-responsive RNAs, RORA and KCTD16 modulate the aggressiveness of CoCl 2 -induced hypoxic osteosarcoma cells

doi: 10.64898/2026.03.30.715387

Figure Lengend Snippet: ( A and I ) Western blots showing RORA, KCTD16, and c-Myc levels in U2OS and MG63 cells, respectively. ( B and J ) Densitometric quantification of the blots using ImageJ demonstrated that overexpression of RORA or KCTD16 led to reduced c-Myc expression in both cell lines. β-Actin was used as a loading control for normalization. ( C and K ) Representative microscopic images of BrdU incorporation assays in RORA and KCTD16 overexpressing U2OS and MG63 cells, respectively. Scale bar = 50 μm. ( D and L ) Quantification of BrdU-positive cells showing significantly decreased proliferative capacity in RORA and KCTD16-overexpressing cells (n ≥ 20 cells per condition). ( E and M ) Representative images of colony formation assays in RORA and KCTD16 overexpressing osteosarcoma cells. ( F and N ) Quantification of colonies demonstrating a marked reduction in clonogenic potential upon RORA or KCTD16 overexpression. ( G and O ) Representative images of migration assays in RORA and KCTD16-overexpressing cells. Scale bar = 50 μm. ( H and P ) Quantification of migrated cells showing significantly impaired migratory capacity in RORA and KCTD16 overexpressing osteosarcoma cells. Statistical analysis was performed using one-way ANOVA, and all data are presented as mean ± SD from three independent biological replicates. Significance is indicated as follows: ns, not significant; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Article Snippet: Human osteosarcoma cell lines U2OS and MG63 were procured from the American Type Culture Collection (ATCC) and the National Centre for Cell Science (NCCS), Pune, respectively.

Techniques: Western Blot, Over Expression, Expressing, Control, BrdU Incorporation Assay, Migration

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin reduces osteosarcoma cell viability and increases membrane permeability. (A) Molecular structure of acetylshikonin. (B) CCK-8 assay results showing the viability of hFOB 1.19 cells following exposure to acetylshikonin (0.5–3 µM) for 24 h (n=4). (C) MG63, (D) HOS and (E) U2OS cell viability was assessed using the CCK-8 assay following treatment with acetylshikonin (0.05–20 µM) for 24 and 48 h (n=4). (F) Phase-contrast microscopy images depicting morphological changes in osteosarcoma cells treated with acetylshikonin (3 µM) for 24 h (n=4). (G) Fluorescence microscopy images showing nuclear staining with Hoechst 33342, membrane integrity with PI and viability with Calcein-AM in osteosarcoma cells treated with acetylshikonin (0.5–10 µM) for 24 h (n=4). Data are presented as the mean ± SD. *P<0.05 vs. untreated control. CCK-8, Cell Counting Kit-8; PI, propidium iodide.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Membrane, Permeability, CCK-8 Assay, Microscopy, Fluorescence, Staining, Control, Cell Counting

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin induces DNA fragmentation in osteosarcoma cells. Osteosarcoma cells (5×10 5 ) were treated with acetylshikonin (0.1–3 µM) for 24 h, then underwent the TUNEL assay. Fluorescence was analyzed by flow cytometry (n=4). Untreated cells were used as controls. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. untreated control.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: TUNEL Assay, Fluorescence, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin induces apoptosis in osteosarcoma cells. Osteosarcoma cells (5×10 5 ) were treated with acetylshikonin (0.1–3 µM) for 24 h, then underwent the Annexin V/PI assay (n=4). Untreated cells were used as controls. Data are presented as the mean ± SD. *P<0.05 vs. untreated control. PI, propidium iodide.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Control

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin promotes cell cycle arrest in osteosarcoma cells. Cell cycle distribution of osteosarcoma cells treated with acetylshikonin (0.1–3 µM) for 24 h, was assessed by propidium iodide staining and flow cytometry. Untreated cells were used as controls. Data are presented as the mean ± SD. *P<0.05 vs. untreated control.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Staining, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin promotes intracellular ROS accumulation. Osteosarcoma cells (5×10 5 ) were treated with acetylshikonin (0.1–3 µM) for 1 h, then stained with 1 µM H 2 DCFDA. Fluorescence was analyzed by flow cytometry (n=4). Untreated cells served as controls. Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. untreated control. ROS, reactive oxygen species.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Staining, Fluorescence, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin disrupts mitochondrial membrane potential. Cells were incubated with acetylshikonin (1 µM) for (A) 2 or (B) 8 h and subsequently stained with JC-1 (n=4). (C) Western blot analysis of Bcl-2, Bcl-xl, Bax and Bak protein expression in osteosarcoma cells following acetylshikonin treatment (0.1–3 µM) for 8 h (n=4). Untreated cells served as controls. Data are presented as the mean ± SD.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Membrane, Incubation, Staining, Western Blot, Expressing

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin decreases mitochondrial volume and enhances lipid peroxidation in osteosarcoma cells. (A) Transmission electron microscopy images of HOS cells treated with acetylshikonin (3 µM) for 24 h, showing a reduction in mitochondrial volume (blue triangles). The red arrow indicates the endoplasmic reticulum. (B) Fluorescence microscopy analysis of lipid peroxidation in osteosarcoma cells treated with acetylshikonin (3 µM) and C11-BODIPY™ 581/591 (n=4). (C-E) Flow cytometric analyses showing lipid peroxidation in osteosarcoma cells incubated with acetylshikonin (0.1–3 µM) and C11-BODIPY (581/591) for 30 min (n=4). (F-H) Intracellular Fe 2+ levels in osteosarcoma cells treated with acetylshikonin (3 µM) for 24 h were quantified using an Fe 2+ detection reagent and microplate reader. (I-K) Western blot analysis of GPX4 protein expression in osteosarcoma cells treated with acetylshikonin (0.1–3 µM) for 8 h (n=4). Untreated cells served as controls. Data are presented as the mean ± SD. *P<0.05 vs. untreated control. GPX4, glutathione peroxidase 4; Fe 2+ , ferrous ion.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Incubation, Western Blot, Expressing, Control

Journal: Molecular Medicine Reports

Article Title: Acetylshikonin induces ferroptosis via the lipid peroxidation pathway in osteosarcoma cells

doi: 10.3892/mmr.2025.13765

Figure Lengend Snippet: Acetylshikonin induces ferroptosis-mediated cell death. (A-C) CCK-8 assay results of osteosarcoma cells pretreated with ferrostatin-1 (10 µM), z-DEVD-FMK (10 µM), necrostatin-1 (10 µM), IM54 (10 µM) and liproxstain-1 (1 µM) for 1 h before exposure to acetylshikonin (3 µM) for 24 h (n=4). (D) Flow cytometric analysis of Annexin V/PI staining in osteosarcoma cells pretreated with ferrostatin-1 (10 µM) for 1 h followed by acetylshikonin (3 µM) for 24 h (n=4). (E) CCK-8 assay results of osteosarcoma cells treated with acetylshikonin (3 µM), erastin (3 µM) and RSL3 (3 µM) for 24 h (n=4). Untreated cells served as controls. Data are presented as the mean ± SD. *P<0.05 vs. untreated control; # P<0.05 vs. acetylshikonin-treated group. CCK-8, Cell Counting Kit-8.

Article Snippet: The human osteosarcoma cell lines (U2OS, HOS and MG63) and normal human osteoblasts (hFOB 1.19) were sourced from the American Type Culture Collection.

Techniques: CCK-8 Assay, Staining, Control, Cell Counting