Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Flow chart depicting candidate gene selection for the DDR and CIN screens. b , Schematic of the design of the DDR and CIN screens. c , Venn diagram showing the six driver genes contributing to DDR and CIN. d , Validation of the six candidate genes by DR-GFP homologous recombination reporter assay; BRCA2 serves as a positive control. HR efficiencies are normalized to those of control samples. Statistical significance was determined by two-sided, one-sample t -test. The data represent means ± s.d. ( n = 3 biological replicates, except for BRCA2, for which n = 2). e , Validation of the six candidate genes by DIvA U2OS-AsiSI site-directed resection assay. Statistical significance was determined by two-sided paired t -test. The data represent means ± s.d. ( n = 3 biological replicates). f , Box plots quantifying RAD51 foci formation in A549 cells following depletion of the six candidate genes, following 6 Gy ionizing irradiation and 1 h of recovery. The box edges represent interquartile ranges, the horizontal lines represent median values and the ranges of the whiskers denote 1.5× the interquartile range ( n = 3 biological replicates; >150 cells quantified per biological replicate). Statistical significance was determined by Kruskal–Wallis test with Dunn’s multiple comparison test. g , Driver mutation distribution and mutational timing of the six candidate genes in the TRACERx 421 cohort. ATM , CHEK2 , ATR , CHEK1 and members of the Fanconi anaemia (FA)/BRCA pathway are included for comparison. FAT1 is highlighted in red. CTRL, control; EGFR, epidermal growth factor receptor; FACS, fluorescence-activated cell sorting; HU, hydroxyurea; IR, ionizing radiation; mut, mutant; nt, nucleotides; WT, wild type.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Selection, Biomarker Discovery, Homologous Recombination, Reporter Assay, Positive Control, Control, Resection Assay, Irradiation, Comparison, Mutagenesis, Fluorescence, FACS

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a-b , Box plots quantifying RAD51 foci in the presence and absence of 6Gy IR in A549 (A) and H1944 (B) cells. Cells were irradiated and allowed 1h recovery. The boxes represent the interquartile range, horizontal lines represent the median and the whiskers range denotes 1.5 x interquartile ranges, n=3 biological replicates, >150 cells quantified per biological replicate, Kruskal-Wallis test with Dunn’s multiple comparison tests. c , Histograms representing cell cycle profiles following siRNA knockdown of 6 candidate DDR+CIN genes in A549 (left) and H1994 (right) cells, using 2 sets of different siRNAs, n=3 biological repeats, 2-way ANOVA with Sidak corrections. d , Driver mutation distribution of FAT1 , and HR-related genes ( ATM-CHEK2 , ATR-CHEK1 , and members of FA/BRCA pathway) in the TRACERx 421 cohort.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Irradiation, Comparison, Knockdown, Mutagenesis

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Top, schematic of dN/dS ratio analysis. Bottom, results of dN/dS ratio analysis in the TRACERx 421 cohort, demonstrating that FAT1 truncation mutations are selected early in LUSC tumour evolution. The data points represent estimated dN/dS ratios and the error bars represent 95% confidence intervals calculated using the genesetdnds function from the R package dNdScv. The TRACERx 421 cohort comprised 233 males and 188 females (421 patients total), corresponding to a 55:45 male:female ratio. 93% of the cohort was from a White ethnic background and the mean age of the patients was 69 years, ranging between 34 and 92 years. Written informed consent was obtained. None of the patients was compensated for their involvement in the study. b , Genomic identification of significant targets In cancer (GISTIC) analysis of LUAD (141 patients) and LUSC tumours (80 patients) in TRACERx with clonal WGD only, demonstrating that SCNA loss at the FAT1 genomic locus ( 4q35.2 ; red text and highlighted) is positively selected in tumours with clonal WGD only. SCNA loci overlapping with common or rare chromosome fragile sites , are annotated (in blue for common fragile sites and in green for rare fragile sites). c , MSAI analysis illustrating that the genomic region of chromosome 4 that harbours the FAT1 gene (arrows) is frequently lost in LUSC. Statistical significance was determined by Fisher’s exact test. In the schematic at the top, paternal and maternal chromosomes are indicated in blue and red, respectively. d , Top, schematic illustrating the location of the FAT1 gene on chromosome 4, together with other 4q35.2 genes within the frequently lost 4q35.2 genomic region. Bottom, selection pressures against losing genes. The data are from the Genome Aggregation Database (gnomAD) and demonstrate high selective pressure against deletion of the FAT1 genomic locus within 4q35.2 . exp, expected; LOF, loss of function; obs, observed.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Selection

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , FAT1 driver mutations are enriched in LUSC tumours in the TRACERx421 LUSC cohort, and mutations are timed early in tumor evolution. b , Graph showing the frequency of FAT1 SCNA loss in the TRACERx 421 cohort. c , GISTIC analysis demonstrating FAT1 genomic loci (4q35.2) SCNA loss is positively selected in both the LUAD and LUSC TRACERx cohort. d , Summary of GISTIC score analysis of LUAD and LUSC TRACERx tumors from Fig. segregated by WGD status. Patients with no WGD and subclonal WGD are included. SCNA loci overlapping with common or rare chromosome fragile sites 90,91 were annotated (common fragile sites=blue, rare fragile sites=green). e , FAT1 promoter hypermethylation is enriched in TRACERx LUSC tumors. Notably, FAT1 promoter hypermethylation also co-occurs with FAT1 SCNA loss. f-g , Correlation between FAT1 promoter hypermethylation and expression levels in LUAD ( f ) and LUSC tumors ( g ).

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Expressing

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Box plots demonstrating the impact of FAT1 siRNA knockdown on early DNA damage signalling and 53BP1 binding in A549 cells. The boxes represent interquartile ranges, the black and red bars represent median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test ( n = 3 biological replicates). The total numbers of cells quantified per condition were as follows: n ≥ 370 (pATM), n ≥ 438 (γH2A.X), n ≥ 448 (53PB1), n ≥ 560 (CtIP) and n ≥ 218 (BRCA1). b , Schematic of the FAT1 functional domains. The full-length FAT1 protein is 4,588 amino acids. c , RAD51 IRIF formation following 6 Gy ionizing radiation and 1 h of recovery in FAT1 CRISPR knockout (sgFAT1) versus control A549 cells with overexpression of HA–FAT1 ICD versus pcDNA3.1. The boxes represent interquartile ranges, the black and red bars represent median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Kruskal–Wallis test followed by Dunn’s test with Bonferroni correction ( n = 3 biological replicates). d – f , Top, cartoons depicting examples of HRD-related large-scale transition (LST; d ), telomeric allelic imbalance (TAI; e ) and LOH ( f ). Bottom left, Permutation analysis showing a correlation between FAT1 CNA and HRD-related genomic signatures based on TCGA LUAD data. Red lines indicates 90 and 95% confidence intervals, blue line indicates observed correlation value. Bottom right, FAT1 driver mutation scores for these respective genetic alterations, based on TRACERx LUAD data. For the TRACERx LUAD data, tumour numbers were as follows: n = 212 (WT) and n = 17 (mut). In the box and whisker plots, the boxes represent interquartile ranges, the lines represent median values and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided mixed-effects linear model with purity as a fixed covariate and tumour ID as a random variable. g , Top, cartoon showing the design of the EJ5–GFP distal end-joining reporter integrated in U2OS cells. Bottom, 53BP1 siRNA knockdown, but not FAT1 knockdown, affects the distal end-joining rate. The data represent means ± s.d. Statistical significance was determined by two-sided repeated measures one-way analysis of variance (ANOVA) with Holm–Šidák correction ( n = 5 biological repeats). h , Top, cartoon showing the design of the EJ2–GFP alternative end-joining reporter integrated into U2OS cells. Bottom, FAT1 siRNA knockdown significantly reduces the alternative end-joining efficiency. The data represent means ± s.d. Statistical significance was determined by two-sided paired t -test ( n = 4 biological repeats). EGF, epidermal growth factor-like domain; LAMG, laminin G-like domain; NLS, nuclear localization signal.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Knockdown, Binding Assay, Functional Assay, CRISPR, Knock-Out, Control, Over Expression, Mutagenesis, Whisker Assay

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Impact of FAT1 on RAD51/γH2A.X foci formation in polyclonal FAT1 wild-type or CRISPR knockout H1944 cells following 6Gy IR. Cells were irradiated and allowed 1 h to recover. Left, quantification showing RAD51 foci in the presence of 6Gy IR. Red-line = mean. The boxes represent the interquartile range, the lines represent the median and the whiskers range denote 1.5 x interquartile ranges, 2-sided Mann-Whitney test, n=3 biological replicates. Right, representative figures illustrating RAD51/γH2A.X foci following 6Gy IR. Bar = 10 μM. Polyclonal FAT1 CRISPR knockout cells were irradiated and allowed 1h recovery. b , Left, representative figures illustrating RAD51 foci in the presence of 6Gy IR. Scale bar =10 µm. Right: box plots showing FAT1 CRISPR knockout A549 cells displayed a reduction in RAD51 foci formation. The boxes represent the interquartile range, the lines represent the median and the whiskers range denotes 1.5 x interquartile ranges, Dunn’s test, n=3 biological replicates. c , Quantification of 53BP1 foci at various time points post 6h IR irradiation in A549 cells. n=2 biological repeats, over 300 cells were quantified in each condition per time point. Solid line = median, dotted lines = quartile ranges, 2-sided Mann-Whitney tests were carried out between two conditions within the same time point. d , Representative western blot showing cellular fractionation of control and FAT1 CRISPR KO A549 cells. Localization of full-length FAT1 protein and C-terminal isoforms were visualized using a FAT1 antibody targeting the C-terminal of the FAT1 protein. Note the loss of FAT1 full-length and C-terminal (p65) signal in FAT1 KO cells. e , A549 FAT1 KO cells were transfected with the HA-FAT1 ICD construct used in Fig. . Top, representative western blot showing the level of endogenous FAT1 and HA-FAT1 ICD construct in A549 cells following 6Gy IR. Levels of KAP1/TRIM28 phosphorylation, γH2A.X, and ubiquitination of γH2A.X are not affected. Bottom, representative figures illustrating RAD51 foci formation in the presence of 6Gy IR, with or without overexpression of HA-FAT1 ICD . Scale bar =10 µm. HD, homozygous deletions; LOH, loss of heterozygosity.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: CRISPR, Knock-Out, Irradiation, MANN-WHITNEY, Western Blot, Cell Fractionation, Control, Transfection, Construct, Phospho-proteomics, Ubiquitin Proteomics, Over Expression

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Comparison, Knockdown, Activity Assay

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , FAT1 knockout exacerbates replication fork stalling in A549 cells. Top, scheme of the nucleotide labeling used to measure replication fork stalling. Bottom (left) quantification; (right), representative image for the DNA fibre experiments. The data represent means ± s.d. Statistical significance was determined by two-sided paired t -test ( n = 3 biological replicates; >600 forks counted in total). Scale bars, 20 µm. b , TCGA LUAD analysis showing that FAT1 copy number loss is significantly correlated with weighted genome instability index measurements. The blue lines indicate FAT1 loss and the red dotted lines indicate the 90 and 95% confidence intervals. Confidence intervals were generated using computational permutation analyses. c , Box plot comparing the numbers of indels in FAT1 WT versus mutated tumours in the Genomics England LUAD and LUSC cohorts. The boxes represent interquartile ranges, the lines represent median values and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test. n = 818 (WT) and n = 16 (mut). d , Transient FAT1 siRNA knockdown induces the formation of 53BP1 bodies in cyclin A-negative U2OS cells following 4 mM hydroxyurea for 5 h and recovery for 24 h. Statistical significance was determined by two-sided Wilcoxon rank-sum test. Scale bars, 10 μm. The red bars in the graph to the left represent mean values ( n = 5 biological replicates). e , f , Transient FAT1 siRNA knockdown in U2OS cells induces the formation of total micronuclei with or without replication stress induced by 5 h of 4 mM hydroxyurea followed by 24 h recovery ( e ), as well as the formation of both acentric and centromeric micronuclei following the hydroxyurea treatment ( f ). The data represent means ± s.d. Statistical significance was determined by one-way ANOVA with Bonferroni correction. Biological repeats: n = 8 ( e ) and n = 4 ( f ). g , h , FAT1 loss elevates the rate of micronuclei formation in response to replication stress induced by 0.2 µM aphidicolin treatment (24 h) following FAT1 CRISPR knockout in A549 cells ( g ) or transient siRNA knockdown in T2P cells ( h ). The data represent means ± s.d. Statistical significance was determined by two-sided Student’s t -test. Biological repeats: n = 4 (total micronuclei in g ), n = 3 (centromeric and acentric micronuclei in g ) and n = 8 ( h ).

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Knock-Out, Labeling, Generated, Knockdown, CRISPR

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Transient FAT1 siRNA knockdown induces the formation of 53BP1 nuclear bodies in T2P cells. The boxes represent the interquartile range, the lines represent the median and the whiskers range denotes 1.5 x interquartile ranges, Dunn’s test, red bar = mean, n=3 biological replicates. Scale bar =10 µm. b , Transient FAT1 siRNA significantly increases the number of lagging chromosomes per cell after replication stress (left, centromeric; right, acentric), Dunn’s test. Over 100 mitotic cells were scored across 3 biological replicates. Red bar = mean. c , Representative metaphase spreads of FAT1 WT and KO A549 cells after 24 hours of aphidicolin treatment. Scale bar = 5µM d , Metaphase chromosome number following transient FAT1 knockdown in H1944 cells. A significant increase in metaphase chromosome number is observed in H1944 cells following FAT1 knockdown. N=3 biological repeats, red bar = mean. 2-sided Welch’s T-test. e , Mitotic error rate in PC9 cells following transient FAT1 knockdown in PC9 cells. one-way ANOVA, N=4 biological repeats, mean±SEM. f , Western blot showing FAT1 knockdown efficiency in PC9 cells. FANCD2 monoubiquitylation and γH2A.X, or expression level of E2F7 are not significantly affected by FAT1 ablation. g , FAT1 knockdown leads to more replication fork stalling in genome-doubled PC9 cells. >600 forks were counted across 3 biological repeats, 2-sided Paired T-test, scale bar =20 µM. h–j , FAT1 knockdown significantly reduces interphase FANCD2 foci ( h , histogram) and mitotic FANCD2 foci ( i and j , histogram and representative image, respectively) despite the increased rate of fork collapse. The boxes represent the interquartile range, black horiziontal bar represent the median and the whiskers range denotes 1.5 x interquartile ranges, red line= mean, N=3 biological replicates, >1200 interphase and >120 mitotic cells scored per condition, Dunn’s test.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Knockdown, Western Blot, Expressing

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Transient FAT1 knockdown significantly increases the mitotic error rate (lagging chromosomes plus DAPI bridges; left; data represent means ± s.d.) and the occurrence of nucleoplasmid bridges (middle; red bars represent mean values) in U2OS cells after 5 h treatment with 4 mM hydroxyurea and 24 h recovery. Statistical significance was determined by one-way ANOVA with Bonferroni correction (left) or Dunn’s test (middle). Right, selected maximum projection images following FAT1 knockdown, showing DAPI-stained mitotic U2OS cells following treatment with 4 mM hydroxyurea and 24 h recovery. Scale bars, 5 μm. Over 100 mitotic cells were scored across three biological replicates. b , Representative PCR-based semi-quantitative DIvA U2OS-AsiSI translocation assay. Transient FAT1 siRNA knockdown increases illegitimate repair products. PCR products generated from the uncut region and the legitimate repair product were used as the loading control. n = 3 biological replicates. c , Histogram (left) and representative images (right) showing that A549 cells with FAT1 loss exhibit a significantly increased number of chromosomal aberrations upon challenge with replication stress induced by 0.2 µM aphidicolin (APH) treatment. Scale bar, 5 μm. The data represent means ± s.d. Statistical significance was determined by one-way ANOVA with Holm–Šidák correction. A total of 60 metaphases were scored across three biological replicates per condition. Blue and red arrows indicate radial chromosomes and chromatid gaps, respectively. d – f , Transient FAT1 siRNA knockdown causes a significant numerical deviation in chromosome number in H1944 cells, as determined by multiple methodologies, including clonal fluorescence in situ hybridization ( d ), ImageStream high-throughput flow cytometry ( e ) and metaphase spreads ( f ). The histogram data represent means ± s.d. For the box plots, the boxes represent interquartile ranges, the black and red lines represent median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test ( d ) or two-sided paired t -test ( e ). n = 3 biological replicates for all cases. bp, base pairs. NT, non-targeting.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Knockdown, Staining, Translocation Assay, Generated, Control, Fluorescence, In Situ Hybridization, High Throughput Screening Assay, Flow Cytometry

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Proportion of FAT1 driver mutations in genome-doubled TRACERx421 tumors. Fisher’s Exact Test, p=0.179. b , FAT1 CRISPR KO leads to elevated MCM7 reloading rate >6N in TP53 mutant PC9 cells, 2-sided Paired T-test, mean±SD, n=3 biological replicates. c , FAT1 ablation leads to elevated MCM7 reloading rate >4N in TP53 WT RPE1 cells with transient FAT1 siRNA transfection, mean±SEM, 2-sided paired T-test, n=3 biological repeat. d , e , Cyclin B1 levels in the G2/M population following CTRL or FAT1 depletion in TP53 WT RPE1 cells. The histogram in d shows no difference in G2/M cyclin B1 levels, mean±SEM, 2-sided paired T-test, n=3 biological repeats. The representative dot plots in e show cyclin B1 levels of the G2 population. f , Nocodazole treatment arrests PC9 cells at mitosis and causes endoreplication (EdU positive cells >6N). FAT1 knockdown does not significantly further increase EdU-positive cells >6N when treated with nocodazole, mean±SEM, 2-sided paired T-Test, n=4 biological replicates. g , Live cell tracking data showing mitotic error rate of RPE1-TERT-FUCCI cells. 2 different FAT1 siRNAs produced a significant increase in mitotic error rate. One-way ANOVA with Sidak correction, mean±SD, n=3 biological repeats. h , Quantification of fixed microscopy images showing that FAT1 siRNA knockdown increases the rate of multinucleated U2OS cells, with or without replication stress induced with hydroxyurea. Left, multinucleation was quantified using phalloidin as a marker for cell boundaries and nuclear envelope stain emerin was used to mark the number of nuclei per cell. Mean±SD, 2-sided paired T-test, Biological repeats: n=4 without damage, n=6 with HU. Right, representative image of cells, scale bar = 10 μM, white arrows = multinucleated cells. i , Quantification of fixed microscopy images showing FAT1 knockdown elevates EdU incorporation rate in multinucleated TP53 KO RPE1-TERT cells, but not in the TP53 WT counterpart. Following aphidicolin-induced replication stress, FAT1 depletion increases EdU incorporation rate in both TP53 WT and TP53 KO cells, mean±SD, one-way ANOVA with Holm-Sidak correction. Biological repeats, no damage n=3; Ctrl siRNA with aphidicolin n=3, FAT1 siRNA with aphidicolin n=4.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: CRISPR, Mutagenesis, Transfection, Knockdown, Cell Tracking Assay, Produced, Microscopy, Marker, Staining

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Representative dot plots demonstrating FAT1 ablation in PC9 cells and assessment of EdU incorporation beyond the normal G2 phase, to visualize WGD. b , Top, representative western blot validating FAT1 knockout. Bottom, quantification of EdU incorporation beyond the normal G2 population showing that FAT1 knockout significantly increases the WGD population in PC9 cells. The data represent means ± s.d. Statistical significance was determined by one-way ANOVA with Bonferroni correction. Biological repeats: n = 7 (sgNT), n = 5 (sgFAT1 clone 1) and n = 4 (sgFAT1 clone 2). c , Schematic (left) and histogram (right) showing the impact of transient FAT1 knockdown in TERT RPE-1 cells on the promotion of WGD through mitotic bypass, as determined by live-cell imaging. The data represent means ± s.e.m. Statistical significance was determined by one-way ANOVA with Bonferroni correction. Biological repeats: n = 3 (with aphidicolin treatment) and n = 6 (without aphidicolin treatment). d , e , Schematics (left) and histograms (right) showing that transient FAT1 siRNA knockdown in TERT RPE-1 cells increases the rates of cytokinesis failure ( d ) and nuclear shape deformation ( e ), as determined by 30× magnification live-cell microscopy imaging at 20 min intervals. The data represent means ± s.e.m. Statistical significance was determined by two-sided paired t -test, At least 200 mitotic events were tracked per condition over five biological replicates. YFP, yellow fluorescent protein.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Western Blot, Knock-Out, Knockdown, Live Cell Imaging, Microscopy, Imaging

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Nuclear morphometric measurements of PC9 cells following CTRL and FAT1 siRNA knockdown. Genome-doubled PC9 cells were treated for 24h with replication stress inducer aphidicolin. Z-project images were segmented and analysed for the difference in nuclear morphology. More than 1680 cells were analysed per condition over 3 biological repeats. 2-sided Mann-Whitney test, p<0.001. b , Live-cell imaging demonstrated higher chromatin bridge formation rate following transient FAT1 siRNA knockdown in RPE1-TERT cells. In addition, the daughter cells are more likely to display nuclear shape deformation morphologies following chromatin bridge formation when FAT1 is depleted. c , Mitotic outcomes of RPE1-TERT cells monitored after initial nuclear shape deformation. Among FAT1 depleted cells, those cells with nuclear shape deformation were less likely to undergo normal mitotic segregation in the following mitosis. 60x magnification live cell microscopy performed using 5 minute intervals, 25 z-steps. scale bar = 5 μM, Chi-squared test. At least 50 mitotic events were tracked per condition over 8 biological repeats. d , Estimation of the effect of FAT1 depletion on mitotic timing in RPE1-TERT-FUCCI cells. At least 100 cells were scored per siRNA knockdown across 3 biological repeats. Cells were imaged at 5-minute intervals over 12 h. Black bar = mean, Kruskal–Wallis test with False Discovery Rate Correction. e , IC90 determination of osimertinib treatment in PC9 cells. f , Experimental flowchart showing the generation of osimertinib-resistant PC9 subclones. g , h , Graphs demonstrating the impact of FAT1 CRISPR KO on the acquisition of osimertinib resistance, as indicated by clone survival ( g ; 2-sided Mann-Whitney Test) and the number of long-term derivation of resistant subclones ( h ; Fisher’s exact test). i , Among Osimertinib-resistant subclones, FAT1 CRISPR KO cells exhibited significantly variable DNA ploidy compared with non-targeting controls. 2-sided Welch’s T-test, non-targeting = 6 clones, FAT1 KO = 31 clones.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Knockdown, MANN-WHITNEY, Live Cell Imaging, Microscopy, CRISPR, Clone Assay

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Western blot demonstrating the impact of FAT1 knockdown on the Src–Mek–Erk signalling axis in hTERT RPE-1 and T2P cells. The results are representative of three repeats. b , Scatter plot showing the impact of transient siRNA depletion of FAT1 on nuclear YAP1 localization using the stringent PTEMF fixation buffer in TERT RPE-1 cells. The red bars represent median values. Statistical significance was determined by two-sided Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Over 170 cells were scored per condition over three biological replicates. c , Top, schematic illustrating the predicted domains of the FAT1 protein and respective regions cloned into a pCMV expression plasmid with an HA epitope tag. Bottom, TEAD activity in FAT1 knockout PC9 hexaploid WGD cells. The normalized TEAD activity was measured as an enrichment of the neonGreen signal over the untransfected background signal in each experiment. The HA signal was used to identify successful FAT1 rescue construct co-transfection at the single-cell level. TEAD activity was elevated in FAT1 -knockout cells but could be further increased by overexpressing the constitutively active HA–YAP1 5SA mutant. Overexpression of the FAT1 wild-type construct repressed TEAD activity. However, overexpression of FAT1 mutants devoid of the MIB2 binding region (mScarlet–HA–FAT1 MIB∆ ) and HA–FAT1 ICD did not repress TEAD activity. The edges of the histograms represent mean values. Statistical significance was determined by two-sided Kruskal–Wallis test followed by Dunn’s multiple comparisons test. More than 95 cells were scored over three biological replicates. d , DSB resection assay, showing that transient knockdown of LATS1 and LATS2 —both negative regulators of YAP1—represses ssDNA formation at DSB break sites (chr22:37194035, ssDNA measured 131 nucleotides from the DSB) in U2OS-AsiSI cells. The data represent means ± s.d. ( n = 4 biological replicates). Statistical significance was determined by two-sided paired t -test. e , Both LATS1 and LATS2 siRNA knockdown in U2OS cells elevate rates of 53BP1 nuclear body formation when challenged with replication stress (5 h of 4 mM hydroxyurea followed by 24 h recovery). The boxes represent interquartile ranges, the black and red lines represent median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Statistical significance was determined by two-sided Wilcoxon rank-sum test. More than 340 cells were scored over three biological replicates. f , Both LATS1 and LATS2 siRNA knockdown in U2OS cells induce centromeric and acentric micronuclei formation following challenge with replication stress (5 h of 4 mM hydroxyurea followed by 24 h recovery), suggestive of a mitotic segregation deficiency. Statistical significance was determined by repeated measures one-way ANOVA. The data represent means ± s.d. ( n = 3 biological replicates). g , Transient siRNA knockdown of LATS1 in U2OS cells elevates the mitotic error rate. The data represent means ± s.d. Statistical significance was determined by repeated measures one-way ANOVA ( n = 3 biological replicates). MIB2, MindBomb2-interacting domain; mS, mScarlet; TM, transmembrane domain.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Western Blot, Knockdown, Clone Assay, Expressing, Plasmid Preparation, Activity Assay, Knock-Out, Construct, Cotransfection, Mutagenesis, Over Expression, Binding Assay, Resection Assay

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a,b Transient siRNA depletion of FAT1 increases the relative YAP1 nuclear localization ratio in RPE1-FUCCI cells ( a ; PTEMF fixation) and T2P cells ( b (left panel); PFA fixation). Scale bars =10 µm. Representative images are shown. The right panel in b shows quantification of the results in T2P cells. Red bar =mean, 2-sided Mann-Whitney test. c , Transient siRNA depletion of FAT1 and LATS1 does not consistently alter the phosphorylation level of YAP1 in RPE1 and T2P cells. d , Plot showing the incidences of amplification and deep deletions of Hippo pathway members in the TRACERx 421 LUSC cohort. The effectors TEAD4 and WWTR1/TAZ are amplified in >10% and >35% of cases, respectively. e , The localization of the HA-epitope tag does not affect the ability to repress TEAD reporter transcriptional activity. Left, Scheme showing the location of the HA-epitope tag in different FAT1 expression constructs. The HA-epitope tag was located either at the N terminus or internally within the FAT1 construct. Right, Histogram showing normalized TEAD transcriptional activity upon overexpression of different FAT1 construct. The edge of histograms denotes the mean values, One-way ANOVA with Holm-Sidak multiple comparisons test, for each condition >95 cells were scored across 3 biological repeats.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: MANN-WHITNEY, Phospho-proteomics, Amplification, Activity Assay, Expressing, Construct, Over Expression

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Representative western blot following LATS1/2 knockdown in the U2OS-AsiSI DIvA system reveals that DNA damage markers such as γH2A.X and phospho-KAP1 are activated proficiently upon damage. b , Middle, representative gel showing that transient knockdown of LATS2 , but not LATS1 , induces an illegitimate repair product in U2OS-AsiSI DIvA cells. PCR products generated from the legitimate repair product were used as the loading control (bottom). n=3 biological repeats. c , Plots of GISTIC scores illustrating that the SCNA loss of the LATS2 genomic loci ( 13q12.11 ) is positively selected in the LUSC but not the LUAD TRACERx421 cohort. Red lines denote q value <0.2. d , e , Histograms representing cell cycle profiles following loss of FAT1 , YAP1 or combined loss of both FAT1 and YAP1 by siRNA in U2OS-AsiSI cells ( d ) and A549 FAT1 WT/KO cells ( e ), n=3 biological repeats, one-way ANOVA with Sidak corrections. f , Histogram quantifying the percentage of FAT1 WT or KO A549 cells with lagging chromosomes after aphidicolin treatment following transient depletion of YAP1 using RNAi, mean±SD, one-way ANOVA with Holm-Sidak multiple correction, biological repeats: FAT1 WT =3, FAT1 KO =4. g , Representative western blot showing YAP1 and FAT1 knockdown efficiency in the RPE1-FUCCI and A549 cells. h , Proposed model showing potential interaction between FAT1 and the Hippo pathway.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Western Blot, Knockdown, Generated, Control

Journal: Nature Cell Biology

Article Title: TRACERx analysis identifies a role for FAT1 in regulating chromosomal instability and whole-genome doubling via Hippo signalling

doi: 10.1038/s41556-024-01558-w

Figure Lengend Snippet: a , Impact of FAT1/YAP1 siRNA co-depletion in U2OS cells, as determined by DR-GFP HR reporter assay. MRE11A siRNA served as a positive control. The HR efficiencies are normalized to those of the control samples. Statistical significance was determined by one-way ANOVA with Holm–Šidák correction. The data represent means ± s.d. Biological replicates: n = 5 (siCTRL and siFAT1), n = 3 (siYAP1) and n = 4 (siFAT1 + siYAP1 and siMRE11A). b , ssDNA resection rates for FAT1 / YAP1 siRNA co-depletion, as determined by DIvA U2OS-AsiSI site-directed resection assay with the DSB site located at chr22:37194035, ssDNA measured 131 nucleotides from the DSB. Statistical significance was determined by one-way ANOVA with Holm–Šidák correction. The data represent means ± s.d. ( n = 3 biological replicates). c , Box plots quantifying RAD51 foci formation in A549 cells following the loss of FAT1 , or the combined loss of both FAT1 and YAP1 , after 6 Gy ionizing irradiation and 1 h recovery. The boxes represent interquartile ranges, the black and red lines represents median and mean values, respectively, and the ranges of the whiskers denote 1.5× the interquartile range. Over 380 cells were scored across three biological replicates. Statistical significance was determined by uncorrected Dunn’s test. d , e , Plots ( d ) and representative images ( e ) illustrating the quantification of mitotic error rates in A549 cells after 24 h of aphidicolin treatment (0.2 µM). FAT1 wild-type or knockout cells were transiently depleted of YAP1 using RNAi. For the mitotic error analysis, statistical significance was determined by one-way ANOVA with Holm–Šidák multiple correction and the data represent means ± s.d. (biological repeats: n = 3 ( FAT1 WT) and n = 4 ( FAT1 knockout)). For the DAPI bridge and Fanconi anaemia complementation group D2 (FANCD2)-flanked DAPI bridge analyses, the red lines represent mean values, the boxes represent interquartile ranges and the ranges of the whiskers denote 1.5× the interquartile range, and statistical significance was determined by Dunn’s test with Bonferroni correction. Over 100 mitotic cells were scored across three biological replicates. Scale bars, 5 µM. f , Results of live-cell imaging analysis, showing that FAT1 / YAP1 double siRNA knockdown in TERT–RPE-1 cells fully rescued the failed cytokinesis and WGD introduced by FAT1 knockdown (left) but only partially ameliorated the nuclear shape deformation (right). Statistical significance was determined by one-way ANOVA. At least five biological replicates were quantified per condition. Biological repeats: n = 4 (siCTRL), n = 7 (siFAT1) and n = 5 (siFAT1 + siYAP1). The data represent means ± s.e.m. g , Histogram illustrating the WGD populations in TP53 wild-type versus knockout RPE-1 cells with or without transient mScarlet–YAP 5SA transfection. The data represent means ± s.e.m. Statistical significance was determined by two-sided Mann–Whitney test ( n = 4 biological repeats). h , Histograms illustrating the total (left) and normalized (right) EdU + WGD populations in FAT1 wild-type versus knockout PC9 cells, with or without transient mScarlet–YAP 5SA transfection. The data represent means ± s.e.m. ( n = 5 biological repeats). Statistical significance was determined by repeated measures one-way ANOVA with Benjamini–Hochberg correction (left) or Friedman test (right). KO, knockout.

Article Snippet: Extended Data Fig. 5 FAT1 KO A549 cells display mild sensitivity towards hydroxyurea, cisplatin and Olaparib. a , Comparison of growth rate trajectories of CTRL, FAT1 and BRCA1 knockdown in HCT116-iRFP cells in the presence and absence of Olaparib. b , Representative images and quantification of clonogenic assays (left: hydroxyurea, replication stress inducer; middle: Olaparib, PARP inhibitor; right: cisplatin, DNA crosslinker). c-d , Genomics England mutational signature analysis showing the signature proportion (left) and signature count (right) of the mutational profile of COSMIC ID6 ( c ) and SBS3 ( d ) mutational signatures, both dependent on indels as a result of end-joining activity.

Techniques: Reporter Assay, Positive Control, Control, Resection Assay, Irradiation, Knock-Out, Live Cell Imaging, Knockdown, Transfection, MANN-WHITNEY

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Affinity-purified immunoprecipitates from HEK293T cells via anti-Flag immunoprecipitation (IP) were analyzed by mass spectrometry to identify unique peptides associated with Flag-tagged FOXN3. The sequences of the PARP1 peptides linked to FOXN3 are presented. ( B ) A co-IP assay was carried out in A549 cells to examine the endogenous association between FOXN3 and PARP1. ( C ) An anti-Flag co-IP assay was performed in A549 cells stably expressing Flag-tagged FOXN3 to assess the association between FOXN3 and PARP1 upon TGF-β treatment (10 ng/ml) at the indicated time points [0, 16, 24, and 48 hours (hr)]. IB, immunoblot. The cells were treated with MG-132 (20 μM, 4 hours) before collection. ( D ) An immunofluorescence assay was performed in A549 cells to assess the colocalization of FOXN3 and PARP1. Scale bar, 10 μm. ( E ) A deletion-mapping assay was performed in HEK293T cells to define the region within FOXN3 associated with PARP1. Flag-tagged FOXN3 WT or its various mutants were cotransfected with HA-tagged PARP1 into HEK293T cells (ΔNTD: Δ1-113aa; ΔForkhead: Δ114-199aa; ΔCTD: Δ200-490aa). ( F ) An in vitro GST pull-down assay was performed to examine the direct interaction between bacterially expressed GST-tagged FOXN3 and His-tagged PARP1 purified from Sf9 insect cells. ( G ) Venn diagrams illustrating the overlapping genes cotargeted by FOXN3 and PARP1 in A549 cells, as determined via CUT&Tag data analysis. ( H ) The normalized distribution of reads for the FOXN3 and PARP1 peaks at the transcription start site (TSS) is presented. ( I ) The binding profiles of FOXN3 and PARP1 at representative Smad response genes in A549 cells were analyzed via CUT&Tag data. The blotting data for (B), (C), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. The data for (C) and (E) were analyzed using one-way ANOVA. ** P < 0.01 and *** P < 0.001.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Affinity Purification, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Mapping Assay, In Vitro, Pull Down Assay, Purification, Binding Assay, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis was conducted to evaluate the levels of PARP1 and FOXN3 in the total lysate of A549 cells treated with TGF-β (10 ng/ml) at the specified time points (0, 16, 24, and 48 hours). TCL, total cell lysate. ( B ) Western blotting measured PARP1 and FOXN3 in the lungs of mice with or without BLM treatment (1.5 mg/kg, 21 days) ( n = 3). ( C ) Western blotting assessed PARP1 and FOXN3 levels in WT and Parp1 −/− A549 cells. ( D ) Western blotting evaluated PARP1 and FOXN3 in the lungs of WT or Parp1 fl/fl mice treated with or without adenovirus-expressed Cre recombinase ( n = 4). ( E ) Western blot analysis was conducted to measure the levels of FOXN3 in Parp1 −/− A549 cells reintroduced with exogenous WT PARP1 or its E988A inactive mutant form. ( F ) Western blot analysis was conducted to measure the levels of FOXN3 in A549 cells treated with BYK204165 (20 μM) or PJ34 (5 μM) at the indicated time points. ( G ) CUT&Tag analyses showing the binding profiles of FOXN3 to Smad response genes in A549 cells with and without BYK204165 treatment (20 μM, 4 hours). ( H ) ChIP assays were performed to detect the distribution of FOXN3 at representative Smad response genes in A549 cells, both with and without BYK204165 treatment (20 μM, 4 hours). ( I ) ChIP assays evaluated Smad protein distribution in WT and Parp1 −/− A549 cells treated with TGF-β (10 ng/ml) for 16 hours. ( J ) Quantitative PCR analyzed Smad response gene expression in WT and Parp1 −/− A549 cells treated with TGF-β (10 ng/ml) for 16 hours. The data in (H) to (J) were assessed by a two-tailed Student’s t test and are shown as the means ± SD. The blotting data for (A), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. Data for (A), (E), and (F) were analyzed using one-way ANOVA, while data for (B) to (D) were assessed using two-tailed Student’s t tests. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Two Tailed Test, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis showing PARP1 protein levels in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment ( n = 3). ( B ) Volcano plot of RNA sequencing data depicting the differential expression of genes in the lungs of mice following conditional KO of PARP1. ( C ) Heatmap analyses of RNA sequencing data showing the changes in the expression of Smad downstream-regulated genes in the lungs of mice following conditional KO of PARP1. ( D ) Histological trichrome staining comparing the fibrotic response in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment. Scale bar, 100 μm. The collagen content from four distinct fields for each sample was quantified using ImageJ software and expressed as a percentage of the total area. ( E ) H&E staining analysis was performed to evaluate the inflammatory response in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment. Scale bar, 100 μm. ( F ) ELISAs were performed to measure TNFα and IL-6 levels in BAL fluid from Parp1 fl/fl mice with and without Cre recombinase treatment following BLM or vehicle control (PBS) treatment ( n = 4). ( G ) Quantitative PCR analysis was performed to assess the RNA levels of profibrotic factors in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment ( n = 4). ( H ) Hydroxyproline measurements comparing the collagen content in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment in the presence or absence of BLM stimulation (1.5 mg/kg, 21 days) ( n = 4). ( I ) Anti-ProSPC and anti–α-SMA immunofluorescence staining of lung sections from Parp1 fl/fl mice with and without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4) was analyzed. Fluorescence intensity from four fields per sample was quantified using ImageJ and expressed as relative intensity. DAPI, 4′,6-diamidino-2-phenylindole. The data for (F) to (H) were assessed using one-way ANOVA, while data for (I) were analyzed using two-tailed Student’s t tests, and all results are presented as the means ± SDs. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using two-tailed Student’s t tests. * P < 0.05.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics, Expressing, Staining, Control, Software, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis of FOXN3 protein levels in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). The mice were treated with or without Cre recombinase. ( B ) Histological trichrome staining assessed lung fibrosis in Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days). Scale bar, 100 μm. ( C ) The collagen content was quantified using ImageJ software for (B). Four distinct fields for each sample were analyzed and expressed as a percentage of the total area. ( D ) H&E staining analysis was performed to evaluate the inflammatory response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days). Scale bar, 100 μm. ( E ) ELISAs were performed to measure the levels of TNFα and IL-6 in BAL fluid from the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( F ) Quantitative PCR analysis was performed to assess the RNA levels of profibrotic factors in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( G ) Hydroxyproline measurements comparing the collagen content in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( H ) Anti-ProSPC and anti–α-SMA immunofluorescence staining analysis of lung sections from BLM-challenged (1.5 mg/kg, 21 days) Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment ( n = 4). Fluorescence intensity from four distinct fields for each sample was quantified using ImageJ software and expressed as the relative fluorescence intensity. The data in (D) to (F) were assessed via one-way ANOVA and are shown as the means ± SD. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using one-way ANOVA. NS means not significant; * P < 0.05 and *** P < 0.001.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, Staining, Software, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A and B ) Western blotting analyzed p38 levels and FOXN3 phosphorylation at S83 and S85 in (A) A549 cells treated with TGF-β (10 ng/ml) over a time course (0, 16, 24, 48 hours) and (B) mouse lungs following BLM administration (1.5 mg/kg) at days 0, 12, and 21. ( C ) Western blotting evaluated p38 levels and FOXN3 phosphorylation in A549 cells treated with or without BYK204165 (20 μM, 4 hours) or PJ34 (5 μM, 4 hours). ( D and E ) Western blot analysis of p38 levels and FOXN3 phosphorylation in A549 cells with or without PARP1 knockdown (D) or KO (E) after TGF-β treatment (10 ng/ml, 16 hours). ( F ) Quantitative PCR detected the RNA levels of p38 in A549 cells treated with or without BYK204165 (20 μM, 4 hours) or PJ34 (5 μM, 4 hours). ( G ) Quantitative PCR detected p38 RNA levels in WT or Parp1 −/− A549 cells treated with TGF-β (10 ng/ml, 16 hours). ( H ) CUT&Tag analyses showing the binding profiles of FOXN3, PARP1, and Smad4 to the p38 promoter in A549 cells treated with or without BYK204165 (20 μM, 4 hours). ( I ) ChIP assays in A549 cells treated with or without BYK204165 (20 μM, 4 hours) assessed FOXN3 and Smad protein binding to the p38 promoter. ( J ) ChIP assays measured FOXN3 and Smad protein binding to the p38 promoter in WT and Parp1 −/− A549 cells after TGF-β treatment (10 ng/ml, 16 hours). ( K ) ChIP assays evaluated Smad protein binding to the p38 promoter in A549 cells with or without FOXN3 knockdown after TGF-β treatment (10 ng/ml, 16 hours). Data from (F), (G), and (I) to (K) were analyzed using two-tailed Student’s t tests and presented as the means ± SD. The blotting data for (A), (C), (D), and (E) were quantified as the means from three independent experiments using ImageJ software. Data for (A), (C), and (D) were analyzed using one-way ANOVA, while data for (E) were assessed using two-tailed Student’s t tests. The blotting data for (B) were also quantified as the means and analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, Phospho-proteomics, Knockdown, Real-time Polymerase Chain Reaction, Binding Assay, Protein Binding, Two Tailed Test, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A and B ) Western blot analysis measured FOXN3 and its S83 and S85 phosphorylation in A549 (A) and NIH3T3 (B) cells with and without p38 siRNA knockdown after TGF-β treatment (10 ng/ml, 16 hours). ( C ) Western blot analysis was conducted to measure the levels of FOXN3 and its S83 and S85 phosphorylation in A549 and NIH3T3 cells treated with SB 203580 (20 μM) at the indicated time points. All the cells were treated with TGF-β (10 ng/ml, 16 hours) before collection. ( D ) ChIP assays were conducted in A549 cells, both with and without p38 knockdown, to evaluate the binding levels of Smad proteins to representative transcriptional targets of Smad proteins. All the cells were treated with TGF-β (10 ng/ml, 16 hours) before collection. ( E ) ChIP assays were conducted in A549 cells, both with and without p38 knockdown, to evaluate the binding levels of FOXN3 to representative transcriptional targets of Smad proteins. All the cells were treated with TGF-β (10 ng/ml, 16 hours) before collection. ( F ) Quantitative PCR analyses were performed in A549 cells, both with and without p38 knockdown, to assess the RNA levels of representative Smad response genes. All the cells were treated with TGF-β (10 ng/ml, 16 hours) before collection. ( G ) Quantitative PCR analyses were performed in NIH3T3 cells, both with and without p38 knockdown, to assess the RNA levels of representative Smad response genes. All the cells were treated with TGF-β (10 ng/ml, 16 hours) before collection. The data in (D) to (G) were assessed via two-tailed Student’s t tests and are shown as the means ± SD. The blotting data for (A) to (C) were representative of three independent experiments and were quantified using ImageJ software. Data for (A) to (C) were analyzed using one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, Phospho-proteomics, Knockdown, Binding Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blotting measured FOXN3 protein levels in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 3). ( B and C ) Trichrome staining analyzed the fibrotic response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration (B). Scale bar, 100 μm. Collagen content was quantified using ImageJ (C), with four fields analyzed per sample, expressed as a percentage of total area. ( D ) H&E staining assessed the inflammatory response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated. Scale bar, 100 μm. ( E ) Quantitative PCR assessed the RNA levels of the profibrotic factors in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). ( F ) Hydroxyproline measurements compared collagen content in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). ( G ) ELISAs measured IL-6 and TNFα levels in BAL fluid from the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). The data in (C) and (E) to (G) were assessed via one-way ANOVA and are shown as the means ± SD. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using one-way ANOVA. NS indicates not significant; * P < 0.05 and ** P < 0.01.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Western Blot, Staining, Real-time Polymerase Chain Reaction, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A and B ) Representative images from the histological immunohistochemical staining analysis showing the expression levels of PARP1 (A) and FOXN3 (B) in the fibrotic lesion areas and the corresponding adjacent normal tissue of patients diagnosed with lung fibrosis. Representative images from four distinct fields for each sample are shown and quantified. Scale bars, 100 μm. ( C ) Results from the quantitative analysis of (A) and (B) are shown as the immunoreactive score (IRS) ( n = 10). ( D ) A proposed working model clarifies the molecular mechanism by which PARP1 regulates the p38-FOXN3 feedback loop to modulate Smad signaling. The schematic representation was created in BioRender. J. Lian (2025); https://biorender.com/p73d358 . The data for (C) was assessed via two-tailed Student’s t tests and are presented as the means ± SD.

Article Snippet: The PARP1 KO A549 cells (#YKO-H954) were purchased from Ubigene Biosciences (Guangzhou, China).

Techniques: Immunohistochemical staining, Staining, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Manganese mediates antiviral effects by driving an ATM-TBK1 phosphorylation signaling pathway

doi: 10.1101/2025.08.20.671272

Figure Lengend Snippet: (A) A549 wild-type and ATM KO A549 cells were treated with increasing concentrations of MnCL 2 . Total cell lysates were collected 24 hours post-treatment using RIPA buffer and analyzed by Western blot using antibodies against ATM, TBK1, and p-TBK1. β-actin served as a loading control. (B) A549 and ATM KO A549 cells were treated with the indicated concentrations of MnCL 2 , followed by stimulation with linearized DNA 24 hours later. After an additional 24 hours, total RNA was extracted, and IFN-λ1 mRNA expression levels were measured by quantitative RT-PCR and normalized to GAPDH. (C–F) 293T cells ( C ), HeLa cells ( D ), MDMs ( E ), and PHA-activated CD4⁺ T cells ( F ) were treated with varying doses of MnCL 2 . Whole cell lysates were collected after 24 hours and analyzed by Western blot using anti-ATM, anti-p-TBK1, and anti-TBK1 antibodies. β-actin was included as an internal loading control.

Article Snippet: A549 cells (ATCC) and ATM knockout (KO) A549 cells (Ubigene, Austin, TX, USA) were cultured in F-12K medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (R&D systems), 25 mM HEPES (Quality Biology), and 5 μg/mL gentamicin (Thermo Fisher Scientific).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Manganese mediates antiviral effects by driving an ATM-TBK1 phosphorylation signaling pathway

doi: 10.1101/2025.08.20.671272

Figure Lengend Snippet: (A) Confocal microscopy analysis of the localization and expression of ATM and p-TBK1 in A549 cells after Mn treatment. A549 cells were grown on 12mm coverslip-inserted 12-well cell culture plates and treated with or without 100 µM MnCL 2 , then fixed, permeabilized and stained with anti-ATM (green), anti-P-TBK1 (Red) antibody at 6, 14 and 24 h after Mn treatment. Nuclei were counterstained with DAPI (blue), and cells were imaged using a 63×/1.4 objective. (B-C) AMINS analysis for the expression of ATM and p-TBK1. A549 cells were seeded on 6-well cell culture plates and treated with or without Mn treatment at 100 µM, then detached using Accutase at 24 h after Mn treatment, fixed, permeabilized, and stained by anti-ATM (Green) and anti-p-TBK1 (Red) antibodies, the single cell morphonology was viewed by bright field. (B) Image galleries display of brightfield, fluorescence dyes AF647 (ATM, channel 11) and AF488 (p-TBK1, channel 2), and composite image of the two fluorescence channels. (C) Histogram overlay of ATM and p-TBK1 intensity of untreated versus Mn-treated focused single cells. The shifted area was shaded in blue, and the shifted percentage (calculated by Fiji) were indicated in the figure.

Article Snippet: A549 cells (ATCC) and ATM knockout (KO) A549 cells (Ubigene, Austin, TX, USA) were cultured in F-12K medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (R&D systems), 25 mM HEPES (Quality Biology), and 5 μg/mL gentamicin (Thermo Fisher Scientific).

Techniques: Confocal Microscopy, Expressing, Cell Culture, Staining, Fluorescence

Journal: bioRxiv

Article Title: Manganese mediates antiviral effects by driving an ATM-TBK1 phosphorylation signaling pathway

doi: 10.1101/2025.08.20.671272

Figure Lengend Snippet: (A) The illustration for Mass spectrometry process and identified phosphorylated sites on ATM by Mn treatment. (B) ATM KO 293T cells were overexpressed by Flag-tagged ATM and His-tagged TBK1 and then treated by Mn at 100 µM. The whole cell lysate was prepared and analyzed by western blot using the antibodies against p-ATM (Ser1981), ATM, p-TBK1 (Ser172), anti-β-actin was included to indicate the loading control (left panel). A densitometry analysis was performed according to the data shown in the left panel by Fiji (right panel). (C) A549 cells were seeded in 6-well cell culture plates and treated with Mn at 100 µM. The total cell lysate was prepared using RIPA buffer at different time points after Mn treatment. The cell lysate was analyzed by anti-p-ATM, anti-ATM, anti-p-TBK1 and TBK1 antibodies. Anti-β-actin was included as the internal loading control for the experiment. A densitometry analysis was performed according to the data shown in left panel by Fiji, the intensity of band for p-ATM or p-TBK1 is normalized by total ATM or TBK1, respectively (right panel).

Article Snippet: A549 cells (ATCC) and ATM knockout (KO) A549 cells (Ubigene, Austin, TX, USA) were cultured in F-12K medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (R&D systems), 25 mM HEPES (Quality Biology), and 5 μg/mL gentamicin (Thermo Fisher Scientific).

Techniques: Mass Spectrometry, Western Blot, Control, Cell Culture