u2os cell lines  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Biosensor-guided discovery of peptide inhibitors targeting the ribosomal protein uS5-PDCD2 chaperone interaction

doi: 10.1016/j.jbc.2026.111415

Figure Lengend Snippet: Inhibition of the uS5-PDCD2 interaction using residues 21 to 50 of uS5. A , Western blot analysis of total extracts (Input, Top ) and anti-Flag purifications (IP:Flag, Bottom ) prepared from HeLa cells that stably expressed Flag-PDCD2 and that were previously transfected with constructs encoding either GFP alone (lane 1), wild-type (WT, lane 2) or F29Y (MT, lane 3) versions of uS5 21-50 -GFP. The blots were analyzed for GFP, Actin, uS5, and Flag-PDCD2. B , quantification of relative uS5 levels copurified from anti-Flag precipitates normalized to Flag-PDCD2 levels. Values were expressed relative to the GFP control vector, which was set to 1.0. The data and error bars represent the average and SD from at least three independent experiments. p -value is indicated, as determined by a one-way ANOVA with Dunnett’s multiple comparisons test. ( C ) U2OS cells that conditionally express Flag-PDCD2 were transfected with either GFP control vector (panels a-d), wild-type (panels e-h) or F29Y (panels i-l) versions of uS5 21-50 -GFP. At the time of transfection, doxycycline was added to the media to induce the expression of Flag-PDCD2. 48 h post-transfection, cells were fixed and simultaneously analysed by direct fluorescence (b, f and j) and immunostaining for Flag-PDCD2 (c, g and k). DNA staining with DAPI shows the nucleus of each cell (a, e and i). Scale bars correspond to 20 μm. D , quantification of nucleus-to-cytoplasmic ratios of Flag-PDCD2 shows a significant decrease in cells that expressed the wild-type version of uS5 21-50 -GFP. More than 60 cells were analyzed for each condition from three independent immunofluorescence experiments. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated. E , Peptide sequences corresponding to residues 21 to 50 of uS5, showing conserved phenylalanines in blue (WT) and substitutions to alanine residues in the mutant in red (MT). F , schematic of the peptide competition experiments using purified uS5-GFP/PDCD2 complex. See text for details. G , Western blot analysis of an uS5-GFP immunoprecipitate that were washed, divided, and treated with increasing concentrations of either wild-type ( Top , WT) or mutant ( Bottom , MT) uS5 21-50 peptides (lanes 2–5) or with no peptide (lane 1). Blots were analyzed for uS5-GFP and endogenous PDCD2. ( H ) Quantification of PDCD2 levels copurified from anti-GFP precipitates normalized to uS5-GFP levels. The values were then set to 1.0 for the control purification in the absence of peptide. Solid lines mark the average binding from three independent replicates, with error bars corresponding to standard deviations. Statistical differences were calculated using a one-way ANOVA with Dunnett’s multiple comparisons test. p -value is indicated.

Article Snippet: Human MOLT-4, HEK293T, HeLa, and U2OS cell lines were acquired from ATCC.

Techniques: Inhibition, Western Blot, Stable Transfection, Transfection, Construct, Control, Plasmid Preparation, Expressing, Fluorescence, Immunostaining, Staining, Immunofluorescence, Mutagenesis, Purification, Binding Assay

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a potent inhibitor of GCN2. A, APL-4098 molecular structure. B, Crystal structure of APL-4098 bound to human GCN2. Backbone of the GCN2 catalytic site (blue ribbon) and binding of APL-4098, with the extensive interaction network in the ATP site shown with dashed red lines (hydrogen bonds) and yellow lines (hydrophobic contacts). C, Biochemical, ADME, and PK profile of APL-4098. *All data generated from a rat intravenous/oral study: 1 mg/kg i.v. and 3 mg/kg orally. 1 Data generated from the i.v. study and 2 data generated from the oral study. D, APL-4098 inhibition of GCN2 kinase activity, measured as the level of eIF2α phosphorylation, using LanthaScreen FRET-based assay. Plot shows mean ± SEM from three representative experiments conducted in duplicate. E, U2OS cells were treated with APL-4098 in the presence or absence of borrelidin as indicated for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from four individual experiments conducted in duplicate. F, U2OS cells were grown in glutamine-depleted medium and treated with APL-4098 as indicated for 4 hours. Cell lysates were analyzed by Western blotting.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Binding Assay, Generated, Inhibition, Activity Assay, Phospho-proteomics, HTRF Assay, Western Blot

Journal: Clinical Cancer Research

Article Title: A Novel Potent and Selective GCN2 Inhibitor, APL-4098, Has Antileukemic Activity through Dysregulation of Mitochondrial Function

doi: 10.1158/1078-0432.CCR-25-1444

Figure Lengend Snippet: APL-4098 is a selective inhibitor of GCN2. A, Eurofins KINOMEScan selectivity panel assay Treespot results for APL-4098 tested at 1 μmol/L. B, Eurofins biochemical K d concentration–response assay. C, U2OS cells were treated with APL-4098 or positive control dabrafenib (bioRxiv 2024.08.14.607626) in the presence of BtdCPU for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments conducted in duplicate. D, U2OS cells were treated with APL-4098 or positive control GSK2606414 in the presence of thapsigargin for 4 hours. Cell lysates were analyzed for phosphorylation of eIF2α using HTRF assay. Plot shows mean ± SEM from three individual experiments.

Article Snippet: The osteosarcoma cell line U2OS (HTB-96, RRID: CVCL_0042) was obtained from ATCC and cultured in DMEM high glucose (Gibco, #41965-039) with 10% FBS (Gibco, #10270-106), 1 mmol/L sodium pyruvate (Gibco, #11360-039), 1% nonessential amino acids (Gibco, #11140-035), and antibiotics (penicillin/streptomycin 100 U/mL and 100 μg/mL; Gibco, #15140-122).

Techniques: Concentration Assay, Positive Control, Phospho-proteomics, HTRF 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 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: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) Representative immunofluorescence images in doxycycline (DOX)-inducible SLFN11-expressing U2OS cells (5 µg/ml DOX, 72 hrs). Without pre-extraction. (B) Schematic overview of the drug screen workflow. (C) Scatter plot showing the drug screen results. Intensity ratio of SLFN11 at 10 µM concentration are shown. For top ranked 7 drugs, intensity ratio of three concentrations are shown (0.1, 1 and 10 µM; see inset). At minimum, 950 cells were analyzed for each condition. (D) Representative immunofluorescence images from the drug screen. Cells were washed with pre-extraction buffer before fixation to only detect chromatin-bound SLFN11 by confocal microscopy (magenta). CPT (camptothecin) and Prexasertib were used as positive controls. Scale bar: 20 µm. (E) Representative immunofluorescence images of a VLX-1570 time-course experiment. Scale bar: 10 µm. DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM) for the indicated times. (F) Quantification of chromatin-bound SLFN11 signals in individual cells for the indicated treatments depicted in panel E (n = 115 – 154 cells per condition). **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). a.u., arbitrary units.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunofluorescence, Expressing, Extraction, Concentration Assay, Confocal Microscopy

Journal: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) SLFN11 chromatin recruitment by VLX-1570 and camptothecin (CPT). Representative immunofluorescence images show chromatin-bound SLFN11 (green), RPA2 (red), and DAPI (blue). DU145 cells were treated with VLX-1570 (1 µM) or CPT (10 µM) for 4 hrs. Scale bar: 10 µm. (B) VLX-1570 recruits SLFN11 in an RPA-independent manner. Left: Representative immunofluorescence images showing chromatin-bound SLFN11 (green), RPA2 (red), and DAPI (blue). Scale bar: 10 µm. DOX-induced SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM) or CPT (10 µM) for 4 hrs. Right: Tracing of the distribution of signals along the white dashed lines shown in the left panels. (C) Representative immunofluorescence images showing chromatin-bound SLFN11 (green), ψH2AX (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM) or CPT (10 µM) for 4 hours. Scale bar: 10 µm. (D, E) Quantification of chromatin-bound SLFN11 and ψH2AX signals in individual cells for the indicated treatments. (n = 118 – 166 cells per condition) *p < 0.05, **p < 0.01 **p < 0.001. ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunofluorescence, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) Left: Representative immunofluorescence images showing the colocalization of chromatin-bound SLFN11 (green) and H3K27ac (red). Nuclei are stained with DAPI (blue). DOX-induced SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM) for 4 hrs. Right: Quantification of chromatin-bound SLFN11 and H3K27ac in individual cells for the indicated treatments (n = 228 – 327 cells per condition). **p < 0.01, ns, non-significant, ***p < 0.001, ****p < 0.0001 (one-way ANOVA). a.u., arbitrary units (B) Left: Representative immunofluorescence images showing SLFN11-dependent inhibition of EU incorporation (green) by VLX-1570. Scale bar: 20 µm. DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM). Nuclei are stained with DAPI (blue). Right: Quantification of EU in individual cells. (n = 109 – 166 cells per condition). **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-way ANOVA) a.u.., arbitrary units (C, D) Pie charts depicting the genomic distribution of SLFN11-Flag (C) and H3K27ac (D) ChIP-seq peaks across annotated genomic features in the presence or absence of VLX-1570. (E, F, and G) Heatmaps and average signal profiles of SLFN11-Flag ChIP-seq (E), H3K27ac ChIP-seq (F), and SLFN11-Flag signal centered on H3K27ac peaks (G) with or without VLX-1570 treatment. (H) Venn diagram showing the number of SLFN11-Flag peaks, H3K27ac peaks, and their overlap identified in the indicated ChIP-seq analyses. 68.4% and 74.1% of the SLFN11 peaks coincide with the H3K27 peaks in the absence and presence of VLX-1570, respectively. (I) Genome browser tracks illustrating ChIP-seq signal enrichment at the FOS promoter for SLFN11-Flag and H3K27ac in the presence or absence of VLX-1570.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunofluorescence, Staining, Expressing, Inhibition, ChIP-sequencing

Journal: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) Representative immunofluorescence images showing chromatin-bound SLFN11 (green) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with PR-619 (10 µM, 4 hrs), CPT (10 µM, 4 hrs), and TAK243 (2 µM, 5 hrs). Scale bar: 5 µm. (B) Quantification of chromatin-bound SLFN11 signals in individual cells for the indicated treatments depicted in panel A (n = 38 – 77 cells per condition). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units. (C) Representative immunofluorescence images showing chromatin-bound SLFN11 (green), Ub (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM, 4 hrs) with or without 1 hr pre-treatment with TAK243 (2 µM). Scale bar: 5 µm. (D) Quantification of chromatin-bound SLFN11 and Ub signals in individual cells for the indicated treatments depicted in panel C (n = 58 – 77 cells per condition). ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units. (E) Representative immunofluorescence images showing unchanged total SLFN11 levels (without pre-extraction; green), Ub (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with same condition as shown in panel C. Scale bar: 10 µm. (F) Quantification of total SLFN11 and Ub signals in individual cells for the indicated treatments depicted in panel E (n = 60 – 81 cells per condition). *p < 0.05, ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunofluorescence, Expressing, Two Tailed Test, Extraction

Journal: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) Immunoprecipitation of RNF168 with SLFN11. HEK293 cells were transfected with Flag-tagged SLFN11 for 48 hrs. Cell lysates were immunoprecipitated with anti-flag M2 agarose beads. RNF168 was probed with anti-RNF168 antibody. (B) Immunoprecipitation of SLFN11 with RNF168. HEK293 cells were transfected with HA-tagged RNF168 for 48 hrs. Cell lysate were immunoprecipitated with anti-HA agarose beads. SLFN11 was probed with anti-SLFN11 antibody. (C) SLFN11 and RNF168 immunoblots of whole-cell lysate showing that SLFN11 expression does not affect RNF168 expression. DOX-inducible SLFN11-expressing U2OS cells were lysed after the DOX induction (72 hrs). (D) Immunoprecipitation of SLFN11 with an antibody against endogenous RNF168. After DOX induction for 72 hrs, U2OS cells were lysed and immunoprecipitated with anti-RNF168 with protein A/G agarose beads. SLFN11 was probed with anti-Flag M2 antibody. (E) Representative immunofluorescence images of proximity ligation assays (PLA) of SLFN11 and RNF168. DOX-inducible SLFN11-expressing U2OS cells were treated for 24 hrs with DOX and then incubated with primary antibodies against SLFN11 and RNF168. (F) Quantification of the PLA as in panel C. Points denote the mean fluorescence intensity (MFI) of PLA/cell with ± SEM (n > 80). ****p < 0.0001 (two-tailed unpaired t test). (G) RNF168-dependent ubiquitylation of SLFN11. HEK293 cells were transfected with His-Ub and siRNF168 mix. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 [SLFN11-(Ub)n] was detected with anti-SLFN11 antibody. (H) Ubiquitylation of SLFN11 by RNF168. 293T cells were transfected with SLFN11-Flag, HA-RNF168, and His-Ub. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was detected with anti-Flag antibody. (I) K27-dependent ubiquitylation of SLFN11 by RNF168. 293T cells were transfected with SLFN11-Flag, HA-RNF168, and His-Ub (WT, K27R, K48R, and K63R). Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was detected with anti-Flag antibody. (J) Mapping of the cellular ubiquitylation sites of SLFN11 by RNF168. Top: schematic representation of the tested lysine residues as potential targets for ubiquitylation by RNF-168 HEK293T cells were transfected with SLFN11-Flag, SLFN11-Flag K/R mutants, HA-RNF168, and His-Ub as indicated. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was probed with anti-Flag antibody. (K) Schematic representation of the location of the ubiquitylated lysine residues of SLFN11 (K390/K391, K429, K743, and K890) mapped onto SLFN11 protein structure (PDB: 7ZEL). (L) Schematic representation of the constructs used for the experiments shown in panel M. (M) Ubiquitylation sites of SLFN11 in its linker domain by RNF168. HEK293T cells were transfected with SLFN11-Flag, SLFN11-Flag K/R mutants (3KR:K390R/K391R/K429R, 2KR: K429R/K743R, as indicated), HA-RNF168, and His-Ub. Cells were lysed and immunoprecipitated with Ni-NTA beads. Ubiquitylated SLFN11 was probed with anti-Flag antibody.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing, Immunofluorescence, Ligation, Incubation, Fluorescence, Two Tailed Test, Construct

Journal: bioRxiv

Article Title: Ubiquitin-dependent recruitment of SLFN11 to chromatin is regulated by deubiquitinase (DUB) and RNF168

doi: 10.64898/2026.03.26.714477

Figure Lengend Snippet: (A) Representative immunofluorescence images showing chromatin-bound SLFN11 (green), RNF168 (red) and DAPI (blue). DOX-inducible SLFN11-expressing U2OS cells were treated with VLX-1570 (1 µM) or CPT (10 µM) for 2 hrs after DOX induction (72 hrs) and siRNF168 transfection (48 hrs). Scale bar: 5 µm. (B, C) Quantification of chromatin-bound SLFN11 and RNF168 signals in individual cells for the indicated treatments (n = 57 – 97 cells per condition). **p < 0.01. ***p < 0.001. ****p < 0.0001 (two-tailed unpaired t test). a.u., arbitrary units. (D) Representative immunofluorescence images showing chromatin-bound SLFN11 (magenta) and DAPI (blue) in DOX-inducible SLFN11 clones (WT, 3KR) of U2OS cells. Cells were treated with VLX-1570 (1 µM) or CPT (10 µM) for 2 hrs. Scale bar: 10 µm. (E) Quantification of chromatin-bound SLFN11 fluorescence (average per condition) for the treatment depicted in panel D (mean ± SEM, N = 411 – 690 cells per condition). ***p < 0.0001 (two-tailed unpaired t test). (F) SLFN11 immunoblots of chromatin fraction. DOX-inducible SLFN11 clones (WT, 3KR) of U2OS cells were treated with VLX-1570 (5 µM, 10 µM, 2 hrs) after the DOX induction (72 hrs), lysed and fractionated with pre-extraction buffer.

Article Snippet: HEK293 (CRL-1573), 293T (CRL-3216), DU145 (HTB-81) and U2OS (HTB-96) cell lines were purchased from American Type Culture Collection (ATCC) and maintained without further authentication.

Techniques: Immunofluorescence, Expressing, Transfection, Two Tailed Test, Clone Assay, Fluorescence, Western Blot, Extraction