human osteosarcoma cell line u2os  (ATCC)

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: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without HU (1 mM, 4 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥242 cells were analyzed per condition). ( B ) Quantification of S9.6 staining in control, C/X3/X2-depleted U2OS cells with or without Aph (0.4 μM, 16 hours) treatment. Data are represented as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥211 cells were analyzed per condition). ( C ) Quantification of γH2AX intensity in the presence or absence of HU (1 mM, 4 hours) treatment in control, C/X3-depleted cells with or without overexpression of RNH1-Flag. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test, ≥241 cells were analyzed per condition). ( D ) Effect of RAD51C or XRCC3 depletion on fork progression/fork symmetry following 50 nM CPT treatment as indicated in the schematic. Top: Experimental schematic; middle: representative images; and bottom: quantification of sister fork ratio. Sister fork ratios were measured by dividing the longer sister fork by the shorter fork (IdU tracts). Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥40 sister fork pairs were analyzed per condition). P values are indicated.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Staining, Control, Two Tailed Test, Over Expression

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Representative immunoblot indicating levels of WT and R258H RAD51C-Flag after depletion of endogenous RAD51C in U2OS cells. MCM3 levels are shown as loading control. ( B ) Quantification of S9.6 staining after transfection of cells with the indicated DNA constructs, as shown in (A). Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥252 cells were analyzed per condition). ( C ) Quantification of RNAPIIS2P + PCNA PLA foci after transfection of cells with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥303 cells were analyzed per condition). ( D ) Representative immunofluorescence images (top) and quantification (bottom) of S9.6 + Flag PLA foci in WT and R258H Flag-RAD51C–transfected cells. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥731 cells were analyzed per condition). Scale bar, 5 μm. ( E ) Co-IP of exogenously expressed Flag-tagged WT and R258H RAD51C, followed by immunoblotting with indicated antibodies. Endogenous protein levels were shown in the input. ( F and G ) Representative immunofluorescence images (F) and quantification (G) of S9.6 + FANCM PLA foci in cells transfected with the indicated DNA constructs. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, two-tailed unpaired t test on means of biological replicates, ≥251 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. res, shRNA-resistant.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Western Blot, Control, Staining, Transfection, Construct, Two Tailed Test, Immunofluorescence, Co-Immunoprecipitation Assay, shRNA

Journal: Science Advances

Article Title: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

doi: 10.1126/sciadv.aea5932

Figure Lengend Snippet: ( A ) Experimental schematic and representative images, and ( B ) quantification of IdU/CldU ratio for fork protection assay in cells expressing the indicated mutants of RAD51C. Data are represented as a scatter plot. Black lines indicate mean values ( n = 3, Mann-Whitney test, ≥203 fibers were analyzed per condition). ( C ) Effect of expressing WT, K131A, and K131R RAD51C mutants on replication restart following the treatment of U2OS cells with 2 mM HU for the indicated time. Top: Experimental schematic; and bottom: quantification of stalled forks. Data show percentage of stalled forks as means ± SD ( n = 3, two-tailed unpaired t test). ( D and E ) Representative immunofluorescence images (D) and quantification (E) of S9.6 staining after transfection of cells with indicated shRNA and RAD51C constructs. Relative nuclear S9.6 intensity data are plotted as a box plot ( n = 3, two-tailed, unpaired t test with Welch’s correction on means of biological replicates, ≥278 cells were analyzed per condition). Scale bar, 5 μm. P values are indicated. EV, empty vector. h, hours.

Article Snippet: Human cell lines U2OS (American Type Culture Collection; HTB-96; RRID: CVCL_0042), HeLa Kyoto (Sachin Kotak laboratory; RRID: CVCL_1922), and hTERT RPE-1 (Sachin Kotak laboratory) were grown in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% Glutamax (Gibco) under standard conditions (5% CO 2 , humidified atmosphere) at 37°C.

Techniques: Expressing, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Staining, Transfection, shRNA, Construct, Plasmid Preparation