Journal: bioRxiv

Article Title: Unlocking DNA Damage Sensitivity of Cancer Cells: The Potential of Splicing Inhibitors

doi: 10.1101/2023.10.08.561421

Figure Lengend Snippet: A – Box plots of tail moments from neutral (double-stranded breaks; on the left) and alkaline (single and double-stranded breaks; on the right)) comet assays of SKOV3 cells after different types of treatment. Tail moment was defined as the product of the tail length and the fraction of total DNA in the tail (Tail moment = tail length x % of DNA in the tail) and was quantified using the CometScore 2.0 software. 60-100 cells were analyzed in each sample. Experiments were performed in triplicate. Asterisks denote statistically significant differences determined using a paired, two-tailed Student’s t-test: * means p < 0.05; ** means p < 0.01. B – Box plots show the number of γH2AX foci per nucleus in SKOV3 cells under different types of treatment. The number of γH2AX foci was calculated using ImageJ software with FindFoci plugins. 60-200 cells were analyzed in each sample. С – Representative immunofluorescence images of SKOV3 cells stained for phosphorylated H2AX (Ser139, green) and with DAPI (blue) after various treatments. Scale bar = 100 μm and 25 μm. D – Bar graph demonstrates the ratio of levels of phosphorylated and total ATM forms according to immunofluorescence staining followed by flow cytometry analysis of SKOV3 cells under different types of treatment. E – Bar graph demonstrates the ratio of levels of phosphorylated and total ATR forms according to immunofluorescence staining followed by flow cytometry analysis of SKOV3 cells under different types of treatment. F – Box plots show the fluorescence intensity of DNApk in SKOV3 cells under different types of treatment. The fluorescence intensity of DNApk was measured using ImageJ software. 60-200 cells were analyzed in each sample. G – Box plots show the fluorescence intensity of R-loops in SKOV3 cells under different types of treatment. The fluorescence intensity of R-loops was measured using ImageJ software. 80-200 cells were analyzed in each sample. H – Box plots show the number of phosphorylated RPA2 foci per nucleus in SKOV3 cells under different types of treatment. The number of phosphorylated RPA2 foci was calculated using ImageJ software with FindFoci plugins. 60-200 cells were analyzed in each sample. I – Cell cycle analysis with flow cytometry of SKOV3 cells stained with DAPI under different types of treatment. Stacked bar graphs show the percentage of cells in different phases of the cell cycle. Percentage of cells in G1, S, and G2/M phases was calculated with NovoExpress software. “Pl-B + CP” - SKOV3 cells were pretreated with 1.56 nM of pladienolide B for 48 hours (in A, D, E) or 6h, 9h, 12h and 24h (in B, C, F, G, H, I), then the medium was changed to the medium with 10 µM of cisplatin for 24 hours; “CP” - SKOV3 cells were incubated with DMSO for 48 hours (in A, D, E) or 6h, 9h, 12h and 24h (in B, C, F, G, H, I), then the medium was changed to the medium with 10 µM of cisplatin for 24 hours (in A, D, E) or 6h, 9h, 12h and 24h (in B, C, F, G, H, I); “Pl-B release” - SKOV3 cells were incubated with 1.56 nM of pladienolide B for 48 hours, then the medium was changed to fresh one without pladienolide B for 24 hours (in A, D, E) or 6h, 9h, 12h and 24h (in B, F, G, H, I); “DMSO” - SKOV3 cells were incubated with DMSO for 48 hours (in A, D, E) or 6h, 9h, 12h and 24h (in F, G); “Pl-B” - SKOV3 cells were treated with 1.56 nM of pladienolide B for 48 hours (in A, D, E). Asterisks denote statistically significant differences determined using Wilcoxon test: ** means p < 0.01 (in B, F, G, H).

Article Snippet: The primary antibodies used were anti-gamma H2A.X (phospho S139) (Sigma-Aldrich, #05-636), anti-RPA32/RPA2 (phospho S33) (Abcam, ab21187), DNA-RNA hybrid (Sigma-Aldrich, MABE1095), and anti-DNA PKcs (phospho S2056) antibody (Abcam, ab18192).

Techniques: Software, Two Tailed Test, Immunofluorescence, Staining, Flow Cytometry, Fluorescence, Cell Cycle Assay, Incubation

Journal: Life Science Alliance

Article Title: FAM111A regulates replication origin activation and cell fitness

doi: 10.26508/lsa.202302111

Figure Lengend Snippet: (A) Quantification of chromatin-bound RPA2 in S phase in pool siRNA-transfected U-2-OS cells. (B) Quantification of chromatin-bound GFP-RPA1 in S phase in siRNA-transfected U-2-OS cells. (C) Immunoblot of whole cell extracts from siRNA-transfected U-2-OS cells. (D) Labeling strategy using BrdU detection under non-denaturing condition to detect single-stranded DNA (ssDNA) exposure. Representative images of ssDNA under unperturbed condition, upon FAM111A OE and after HCl treatment. HCl is used as a positive control for BrdU incorporation. (E) Quantification of ssDNA in proliferating cell nuclear antigen (PCNA)-positive cells treated with 300 nM UCN-01 for 1 h. n > 794 per condition. (F) Quantification of RPA in PCNA-positive cells treated with HU, camptothecin or Bleo, analyzed as in . For (A, B, C), cells were treated with 3 mM HU for 2 h. For (A, B), S phase cells were gated based on chromatin-bound PCNA intensities. (A, B, C, D, E, F) Data are representative of three (A, B, C, D) and two (E, F) independent experiments. (A, B, E, F) , Mann–Whitney test, *** P < 0.001, * P < 0.05, n.s., non-significant. Source data are available for this figure.

Article Snippet: The following antibodies were used: FAM111A (HPA040176, 1:500–1:1,000; Sigma-Aldrich, ab184572, 1:500–1:1,000; Abcam), PCNA (ab29, clone PC10, 1:1,000; ab18197, 1:1,000 for immunofluorescence; Abcam), H4K12ac (07-595, 1/1,000; Millipore), histone H3 (ab10799, 1:1,000; Abcam), MCM3 (ab4460, 1:1,000; Abcam), MCM2 (610701; 1:1,000; BD Bioscience, ab4461 1:1,000 for immunofluorescence; Abcam), CDC45 (CST #11881, 1:1,000), RPA2 (ab2175; clone 9H8, 1:1,000, 1:300 for immunofluorescence), histone H4 (05-858, 1:1,000; Millipore), GINS1 (1:500; kind gift from Karim Labib’s lab), p-CHK1 S345 (CST #2348; 1:1,000), CHK1 (sc-56291, 1:1,000; Santa Cruz), p-CHK2 T68 (CST #2661, 1:1,000), p-RPA S4/S8 (A300-245A-M 1:1,000; Bethyl), p-H2A.X S139 (γH2A.X) (CST #2577, 1:1,000, 1:500 for immunofluorescence), p-RPA S33 (A300-246A, 1:1,000; Bethyl), anti-HA (CST #3724, 1:1,000; BioLegend 901501, 1:000 for immunofluorescence), GAPDH (CST #2118, 1:1,000), CDC45 (CST #11881, 1/50), FAM111B (PA5-58474, 1:1,000–1:2,000; Invitrogen).

Techniques: Transfection, Western Blot, Labeling, Positive Control, BrdU Incorporation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

doi: 10.1101/2023.09.15.556420

Figure Lengend Snippet: ( A ) Model of CFTR-F508del folding, correction, and triage. Sequential CFTR-F508del folding states are in equilibrium at the ER and converting between states is increased by corrector binding. The effect of the F508del mutation on the equilibrium between core-glycosylated states B 1 -B 3 is indicated by the thickness of forward and reverse arrows. B 1 : Nascent CFTR-F508del with unstructured NBD1. This folding state is targeted by ERAD and is incapable of exiting the ER. B 2 : more native-like folding state that is not an ERAD substrate. Binding of tezacaftor to B 2 lowers probability of converting back to B 1 . B 3 : later folding state that is not an ERAD substrate and has the capacity to bind to elexacaftor, which slows the rate of conversion back to B 2 . B 3 is near-native with a structured NBD1 and can exit the ER. C: complex-glycosylated folding-state in post-ER compartments. While binding of correctors changes the equilibrium constants between states, knocking out ERAD components does not. Structural cartoons are adapted from . ( B-E ) Inhibiting ERAD increases the amount of mNG-F508del available for correction. Cells were administered tezacaftor (VX-661, 5 μM) and/or elexacaftor (VX-445, 5 μM) for 16 hours. Elexacaftor and tezacaftor synergistically increase mNG-F508del half-life ( B ) and maturation ( C ). Knocking out RNF5 increased the effect of ET on mNG-F508del half-life ( D ) and maturation ( E ). Methods and quantification are as in .

Article Snippet: Antibodies used in this study were CFTR M3A7 Mouse Monoclonal (Millipore Sigma, MAB3480, 1:1000), CFTR L12B4 Mouse Monoclonal (Millipore Sigma, MAB3484, 1:1000), CFTR 570 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A2, 1:1000), CFTR 596 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A4,1:1000), GAPDH 14C10 Rabbit Monoclonal (Cell Signaling Technologies, 2118S, 1:5000), RNF5 22B3 Mouse Monoclonal (Santa Cruz Biotechnology, sc-81716, 1:1000), RNF185 + RNF5 Rabbit Monoclonal (Abcam, ab181999, 1:1000), and ɑ-Tubulin Rabbit Polyclonal (Abcam, ab15246, 1:1000-2000),

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

doi: 10.1101/2023.09.15.556420

Figure Lengend Snippet: ( A ) Schematic of genome-wide screening method. ( B ) Volcano plots of single knockout CRISPR screen. casTLE analysis of genome-wide screens with mNG-F508del (n = 2 biological replicates). Grey = genes observed in screens. Black = significant genes (FDR < 1%). Red = significant E3 ubiquitin ligases and adaptors. Blue = significant E2 ubiquitin conjugating enzymes. Green = significant E1 ubiquitin activating enzymes. Pink = significant chaperones and co-chaperones. See for doxycycline washout curves for screens, for correlation between replicates, and Table S1 for complete casTLE analysis. ( C-F ) Validation of RNF5 and UBE2D3 hits. Pooled knockout mNG-F508del cell lines were generated using Cas9 RNP nucleofection with three sgRNAs per gene. sgControl is a control guide targeting the AAVS1 safe-harbor locus (* = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). ( C ) Effect of gene disruption on mNG-F508del degradation kinetics, as in . ( D ) Quantification of data from ( C ). (Left) fold change in steady-state MFI at t = 0 prior to the addition of emetine. (Center) half-life and (right) plateau as in & . ( E ) Immunoblot of mNG-F508del expression and knockout efficiency as in . ( F ) Quantification of data from (n = 3 biological replicates imaged on the same immunoblot).

Article Snippet: Antibodies used in this study were CFTR M3A7 Mouse Monoclonal (Millipore Sigma, MAB3480, 1:1000), CFTR L12B4 Mouse Monoclonal (Millipore Sigma, MAB3484, 1:1000), CFTR 570 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A2, 1:1000), CFTR 596 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A4,1:1000), GAPDH 14C10 Rabbit Monoclonal (Cell Signaling Technologies, 2118S, 1:5000), RNF5 22B3 Mouse Monoclonal (Santa Cruz Biotechnology, sc-81716, 1:1000), RNF185 + RNF5 Rabbit Monoclonal (Abcam, ab181999, 1:1000), and ɑ-Tubulin Rabbit Polyclonal (Abcam, ab15246, 1:1000-2000),

Techniques: Genome Wide, Knock-Out, CRISPR, Generated, Disruption, Western Blot, Expressing

Journal: bioRxiv

Article Title: Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

doi: 10.1101/2023.09.15.556420

Figure Lengend Snippet: . ( A ) Doxycycline washout curve for genome-wide screens overlaid with a translational shutoff curve. Translational inhibition with emetine and dox washouts were conducted 12 hours post induction with 0.1 μg/ml dox. (n = 4 spinner flasks per doxycycline washout screen, error bars = SD, n = 1 for translational shutoff). ( B ) Correlation between casTLE scores from screen replicates. Curve = linear regression. Grey = genes observed in screens. Black = significant genes (FDR < 1%). ( C ) Volcano plot of casTLE analysis of single knockout CRISPR screens. Data as in . Purple = significant mRNA catabolic process (GO:0006402) genes. Grey = genes observed in screens. Black = significant genes (FDR < 1%). ( D ) Knocking out the gene encoding the UFMylation activating enzyme, UBA5 , does not increase mNG-F508del stability. Pooled UBA5 KO reporter cell lines generated using lentiviral integration and puromycin selection of guide RNA transgenes in mNG-F508del reporter cells expressing Cas9-BFP. sgControl = a safe-targeting negative control guide RNA selected from the Bassik Lab Human CRISPR-Cas9 Deletion Library (sgSAFE.6665). Knockout efficiency of sgUBA5 validated in . Methods and quantification are as in . (*** = p < 0.001, **** = p < 0.0001, unpaired t-test). ( E ) Knocking out UBA5 increases mNG-F508del transcript abundance. qPCR analysis of UBA5 KO reporter cell lines. Relative fold changes were calculated using the 2 -ΔΔCt method with GAPDH as the reference gene (n = 3 technical replicates, **** = p < 0.0001, unpaired t-test). ( F ) The casTLE gene effects and scores are lower for mNG-F508del CRISPR knockout screens than those of model ERAD substrates. Data replotted from . Grey = genes observed in screens. Black = significant genes (FDR < 1%). Red = significant E3 ligases. Blue = significant E2 conjugating enzymes. ( G ) Knocking out RNF5 and UBE2D3 singly or in combination have modest effects on mNG-WT degradation. Pooled single and double knockout mNG-WT Cas9-BFP reporter cell lines generated using lentiviral integration and puromycin selection of dual-guide RNA transgenes. sgControl = a dual guide construct expressing two safe-targeting negative control guide RNAs (sgSAFE.6665). Degradation kinetics methods and quantification are the same as in .

Article Snippet: Antibodies used in this study were CFTR M3A7 Mouse Monoclonal (Millipore Sigma, MAB3480, 1:1000), CFTR L12B4 Mouse Monoclonal (Millipore Sigma, MAB3484, 1:1000), CFTR 570 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A2, 1:1000), CFTR 596 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A4,1:1000), GAPDH 14C10 Rabbit Monoclonal (Cell Signaling Technologies, 2118S, 1:5000), RNF5 22B3 Mouse Monoclonal (Santa Cruz Biotechnology, sc-81716, 1:1000), RNF185 + RNF5 Rabbit Monoclonal (Abcam, ab181999, 1:1000), and ɑ-Tubulin Rabbit Polyclonal (Abcam, ab15246, 1:1000-2000),

Techniques: Genome Wide, Inhibition, Knock-Out, CRISPR, Generated, Selection, Expressing, Negative Control, Double Knockout, Construct

Journal: bioRxiv

Article Title: Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

doi: 10.1101/2023.09.15.556420

Figure Lengend Snippet: ( A ) casTLE analysis of sensitized screens with the UBAL sublibrary. Grey = genes observed in screen. Black = significant genes (FDR < 1%). Red = significant E3 ubiquitin ligases. Blue = significant E2 ubiquitin conjugating enzymes. Green = significant E1 ubiquitin activating enzymes. n = 2 biological replicates per genotype. ( B-D ) Knocking out RNF185 and RNF5 simultaneously has a synergistic effect on mNG-F508del half-life ( B ) but does not increase maturation ( C ). ( D ) mNG-F508del has an equivalent half-life in RNF185/UBE2D3 KO and UBE2D3 KO backgrounds. Methods and analyses are the same as in . ( E ) Inhibiting ubiquitylation does not increase mNG-F508del maturation. Immunoblots of mNG-CFTR-F508del cells treated with 2 μM TAK-243 for 3 hours after dox induction. Methods and analyses are the same as in .

Article Snippet: Antibodies used in this study were CFTR M3A7 Mouse Monoclonal (Millipore Sigma, MAB3480, 1:1000), CFTR L12B4 Mouse Monoclonal (Millipore Sigma, MAB3484, 1:1000), CFTR 570 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A2, 1:1000), CFTR 596 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A4,1:1000), GAPDH 14C10 Rabbit Monoclonal (Cell Signaling Technologies, 2118S, 1:5000), RNF5 22B3 Mouse Monoclonal (Santa Cruz Biotechnology, sc-81716, 1:1000), RNF185 + RNF5 Rabbit Monoclonal (Abcam, ab181999, 1:1000), and ɑ-Tubulin Rabbit Polyclonal (Abcam, ab15246, 1:1000-2000),

Techniques: Western Blot

Journal: bioRxiv

Article Title: Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states

doi: 10.1101/2023.09.15.556420

Figure Lengend Snippet: . ( A-H ) Knocking out RNF5 or UBE2D3 has an additive effect with tezacaftor and elexacaftor on mNG-F508del half-life ( A , C , E , F , G ) and maturation ( B , D , H ). Cells were administered tezacaftor (VX-661, 5 μM) and/or elexacaftor (VX-445, 5 μM) for 16 hours. Methods and quantification are as in .

Article Snippet: Antibodies used in this study were CFTR M3A7 Mouse Monoclonal (Millipore Sigma, MAB3480, 1:1000), CFTR L12B4 Mouse Monoclonal (Millipore Sigma, MAB3484, 1:1000), CFTR 570 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A2, 1:1000), CFTR 596 Mouse Monoclonal (CFF-UNC CFTR Antibody Distribution Program, A4,1:1000), GAPDH 14C10 Rabbit Monoclonal (Cell Signaling Technologies, 2118S, 1:5000), RNF5 22B3 Mouse Monoclonal (Santa Cruz Biotechnology, sc-81716, 1:1000), RNF185 + RNF5 Rabbit Monoclonal (Abcam, ab181999, 1:1000), and ɑ-Tubulin Rabbit Polyclonal (Abcam, ab15246, 1:1000-2000),

Techniques:

Journal: bioRxiv

Article Title: UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells

doi: 10.1101/2023.09.04.556280

Figure Lengend Snippet: (A) Representative images of pRPA S33 (green) and RPA (magenta) in U2OS and FBH1 KO cells treated with 2mM HU +/- B02 for 24 hours. DNA is stained with DAPI (blue). Scale bar =100µm. Graph represents mean of three independent experiments. Error bars are standard deviation. Scale bar =100µm. (B) Representative images of pRPA S4/8 (green) and RPA (magenta) in U2OS and FBH1 KO cells treated with 2mM HU +/- B02 for 24 hours. DNA is stained with DAPI (blue). Scale bar =100µm. Graph represents mean of three independent experiments. Error bars are standard deviation. (C) Representative images of γ−H2AX (magenta) and PCNA (green) in U2OS and FBH1 KO cells treated with 2mM HU +/- B02 for 24 hours. DNA is stained with DAPI (blue) Scale bar =100µm. Graph represents mean of three independent experiments. Error bars are standard deviation. ns-not significant * p <0.05, ** p <0.005, Two-way ANOVA, Tukey.

Article Snippet: The primary antibodies used were Mouse anti-RPA32/RPA2 [9H8] (Abcam Ab2175, 1:1000), Rabbit phospho-RPA32 (Ser33) polyclonal (Bethyl Laboratories, A300-246A-T,1:1000), Rabbit phospho-RPA32 (Ser4, Ser8) polyclonal (Bethyl Laboratories, A300-245A-M, 1:1000), Rabbit anti-PCNA (Abcam, Ab18197, 1:1000), and Mouse anti-gamma H2AX (phospho S139) [9F3] (Abcam, Ab26350, 1:1000).

Techniques: Staining, Standard Deviation

Journal: bioRxiv

Article Title: UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells

doi: 10.1101/2023.09.04.556280

Figure Lengend Snippet: (A) Representative images of pRPA S4/8 (green) and RPA (magenta) in U2OS and FBH1 KO cells treated with 2mM HU for 24 hours. DNA is stained with DAPI (blue). Scale bar = 100µm. Graph represents mean of four independent experiments. Error bars are standard deviation. (B) Representative images of γ-H2AX (green) and PCNA (magenta) in U2OS and FBH1 KO cells treated with 2mM HU for 24 hours. DNA is stained with DAPI (blue). Scale bar =100µm. Graph represents mean of four independent experiments. Error bars are standard deviation. ns – not significant. ***p<0.0005, **** p<0.0001, Two-way ANOVA, Tukey.

Article Snippet: The primary antibodies used were Mouse anti-RPA32/RPA2 [9H8] (Abcam Ab2175, 1:1000), Rabbit phospho-RPA32 (Ser33) polyclonal (Bethyl Laboratories, A300-246A-T,1:1000), Rabbit phospho-RPA32 (Ser4, Ser8) polyclonal (Bethyl Laboratories, A300-245A-M, 1:1000), Rabbit anti-PCNA (Abcam, Ab18197, 1:1000), and Mouse anti-gamma H2AX (phospho S139) [9F3] (Abcam, Ab26350, 1:1000).

Techniques: Staining, Standard Deviation

Journal: bioRxiv

Article Title: UBC13-mediated template switching promotes replication stress resistance in FBH1-deficient cells

doi: 10.1101/2023.09.04.556280

Figure Lengend Snippet: (A) Representative images of pRPA S4/8 (green) and RPA (magenta) in RPE-1 cells treated with 2mM HU after transfection with the indicated siRNAs. DNA is stained with DAPI (blue). Scale bar =100µm. Graph represents mean of three independent experiments. Error bars are standard deviation. (B) Representative images of γ-H2AX (green) and PCNA (magenta) in RPE-1 cells treated with 2mM HU after transfection with the indicated siRNAs. DNA is stained with DAPI (blue) Scale bar =100µm. Graph represents mean of three independent experiments. Error bars are standard deviation. ns – not significant. **** - p<0.0001. Two-way ANOVA, Tukey.

Article Snippet: The primary antibodies used were Mouse anti-RPA32/RPA2 [9H8] (Abcam Ab2175, 1:1000), Rabbit phospho-RPA32 (Ser33) polyclonal (Bethyl Laboratories, A300-246A-T,1:1000), Rabbit phospho-RPA32 (Ser4, Ser8) polyclonal (Bethyl Laboratories, A300-245A-M, 1:1000), Rabbit anti-PCNA (Abcam, Ab18197, 1:1000), and Mouse anti-gamma H2AX (phospho S139) [9F3] (Abcam, Ab26350, 1:1000).

Techniques: Transfection, Staining, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: β-Catenin and SOX2 Interaction Regulate Visual Experience-Dependent Cell Homeostasis in the Developing Xenopus Thalamus

doi: 10.3390/ijms241713593

Figure Lengend Snippet: List of antibodies.

Article Snippet: PCNA, rabbit , Recombinant human PCNA , Abcam , ab18197 , AB_444313 , 1:200 (IF).

Techniques: Recombinant

Journal: International Journal of Molecular Sciences

Article Title: β-Catenin and SOX2 Interaction Regulate Visual Experience-Dependent Cell Homeostasis in the Developing Xenopus Thalamus

doi: 10.3390/ijms241713593

Figure Lengend Snippet: List of antibodies.

Article Snippet: PCNA, rabbit , Recombinant human PCNA , Abcam , ab18197 , AB_444313 , 1:200 (IF).

Techniques: Recombinant

Journal: Nucleic Acids Research

Article Title: DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

doi: 10.1093/nar/gkad549

Figure Lengend Snippet: SIRT2 interacts in a complex with DNA-PKcs. ( A ) SIRT2 was IP’ed from HCT116 cells and IP’ed lysates were subjected to mass spectrometry analysis to identify SIRT2-interacting proteins. Total (T) and unique (U) number of peptides pulled-down for each IP’ed protein was measured and DNA-PKcs was identified as one of the top SIRT2-interactors. IP and mass spectrometry were conducted in duplicate (SIRT2 IP-1 and SIRT2 IP-2) with comparable results. IP with IgG (Control) was included. ( B ) Immunoprecipitation (IP) of endogenous DNA-PKcs or IgG was carried out on HeLa whole cell lysates, run on SDS-PAGE and immunoblotted for DNA-PKcs and SIRT2. The adjoining input is seen directly adjacent to the IP. ( C ) IP of endogenous SIRT2 or IgG was carried out on HeLa whole cell lysates with or without ethidium bromide (EtBr 50 μg/ml) or IR treatment (0 or 10 Gy IR), run on SDS-PAGE and immunoblotted for DNA-PKcs and SIRT2. The adjoining input is seen directly below the IP. ( D ) IP of overexpressed Ku80-GFP or IgG was carried out on HeLa whole cell lysates, run on SDS-PAGE and immunoblotted for DNA-PKcs, SIRT2, and GFP. The adjoining input is seen directly below the IP.

Article Snippet: Cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05–636), DNA-PKcs phosphoserine 2056 (Abcam, ab18192), anti-FLAG (Cell Signaling: 2368P), Artemis pS516 (Abcam, ab138411) or XRCC4 pS260 (Sigma, HPA006801) primary antibodies, followed by Alexa Fluor 555/488 secondary antibodies and DAPI.

Techniques: Mass Spectrometry, Immunoprecipitation, SDS Page

Journal: Nucleic Acids Research

Article Title: DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

doi: 10.1093/nar/gkad549

Figure Lengend Snippet: SIRT2 deacetylates DNA-PKcs in response to DNA damage. ( A ) In vitro deacetylation assay of DNA-PKcs by SIRT2. Acetylated endogenous DNA-PKcs was immunopurified from HeLa cells treated overnight with TSA and nicotinamide. SIRT2-FLAG WT and SIRT2-FLAG H187Y proteins were purified from 293T cells and eluted from beads. Acetylated DNA-PKcs was incubated in an in vitro deacetylation reaction with SIRT2-FLAG WT or SIRT2-FLAG H187Y with or without NAD+. Nicotinamide was also added, where indicated, as an inhibitor of SIRT2. ( B , C ) Cellular deacetylation assay. HeLa cells were transfected with SIRT2-FLAG WT, H187Y, or mock transfected. Cells received overnight TSA treatment with or without nicotinamide as indicated. Cells were also treated with or without 10 Gy IR. Endogenous DNA-PKcs or IgG was IP’d from whole cell lysate. The resulting western blots for IP and input were immunoblotted with DNA-PKcs, pan-acetyl, FLAG, and GAPDH antibodies. Shown are a representative western blot (B) and quantification of average and SD of co-IP’ed acetylated DNA-PKcs from three independent experiments (C). Acetylated DNA-PKcs was quantified using the Image Studio software and is expressed relative to the acetylated DNA-PKcs in the no treatment control sample, after normalization to total DNA-PKcs IP’ed. ** P < 0.01, *** P < 0.001. ( D , E) Endogenous DNA-PKcs acetylation before and after damage and before and after SIRT2 knockdown was assessed through IP of DNA-PKcs or IgG from HeLa cells. HeLa cells were transfected with or without NT siRNA or SIRT2 siRNA. The resulting blots were stained for pan-acetyl, DNA-PKcs, SIRT2, and/or GAPDH. A representative western blot (D) and quantification of average and SD of co-IP’ed acetylated DNA-PKcs from three independent experiments (E) are shown. Acetylated DNA-PKcs was calculated as described in (C). *** P < 0.001. ( F ) HeLa cells were transfected with or without SIRT6-FLAG WT. Endogenous DNA-PKcs or IgG was IP’d from whole cell lysate and run on SDS-PAGE gel. The resulting western blots for IP and input were immunoblotted for DNA-PKcs, pan-acetyl, FLAG and GAPDH.

Article Snippet: Cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05–636), DNA-PKcs phosphoserine 2056 (Abcam, ab18192), anti-FLAG (Cell Signaling: 2368P), Artemis pS516 (Abcam, ab138411) or XRCC4 pS260 (Sigma, HPA006801) primary antibodies, followed by Alexa Fluor 555/488 secondary antibodies and DAPI.

Techniques: In Vitro, Purification, Incubation, Transfection, Western Blot, Software, Staining, SDS Page

Journal: Nucleic Acids Research

Article Title: DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

doi: 10.1093/nar/gkad549

Figure Lengend Snippet: SIRT2 deacetylase activity promotes DNA-PKcs localization to DNA damage sites and interaction with Ku. ( A ) DNA-PKcs-GFP stably transfected U2OS cells were subjected to laser microirradiation following transfection with SIRT2 or NT siRNA. The laser output was set to 75%, which can reproducibly give focused DNA-PKcs-GFP stripes. ( B ) Western blot demonstrating SIRT2 knockdown generated from cells used in (A). ( C ) DNA-PKcs-GFP stably transfected CHO V3 cells were subjected to laser microirradiation with and without SIRT2 inhibition by SirReal2 (50 uM) or DMSO. ( D ) CHO V3 cells treated with or without SirReal2 or DMSO were fixed 2 min post microirradiation and stained for γH2AX in red, DNA-PKcs-GFP in green, and DAPI stain in the overlay. ( E ) Fluorescence of the GFP stripes was measured for part A, recorded at indicated time points, and analyzed for quantitation using Image Studio Lite Software. The average of 15 biological replicates at each time point per condition was plotted. Error bars represent standard deviation: NS indicates P ≥ 0.05, * P < 0.05, ** P < 0.01. ( F ) Quantitation of DNA-PKcs-GFP localization to sites of damage was performed for 15 biological replicates per condition. Fluorescence of the GFP stripes were measured for part C, recorded at indicated time points, and analyzed for quantitation using Image Studio Lite Software. The average of each time point's replicates was plotted. Error bars represent standard deviation. NS indicates P ≥ 0.05, * P < 0.05, ** P < 0.01. ( G ) WT and SIRT2 KO HCT116 cells were transfected with and without Ku70-GFP and treated with or without 10 Gy IR. Ku70-GFP was IP’d, run on SDS-PAGE gels, and immunoblotted for DNA-PKcs, GFP, SIRT2, and GAPDH. DNA-PKcs pulled down with Ku70-GFP was quantified (values shown under the DNA-PKcs IP blot) using the Image Studio software and is shown as a fraction of DNA-PKcs co-IP’ed under control conditions, after normalization to IP’ed Ku70-GFP. ( H ) SIRT2 KO HCT116 cells were transfected with and without Ku70-GFP, SIRT2-FLAG WT, and H187Y and treated with or without 10 Gy IR. Ku70-GFP was IP’d, run on SDS-PAGE gel, and, immunoblotted for DNA-PKcs, GFP, SIRT2, and GAPDH. DNA-PKcs co-IP’ed with Ku70-GFP was quantified as described in (G).

Article Snippet: Cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05–636), DNA-PKcs phosphoserine 2056 (Abcam, ab18192), anti-FLAG (Cell Signaling: 2368P), Artemis pS516 (Abcam, ab138411) or XRCC4 pS260 (Sigma, HPA006801) primary antibodies, followed by Alexa Fluor 555/488 secondary antibodies and DAPI.

Techniques: Histone Deacetylase Assay, Activity Assay, Stable Transfection, Transfection, Western Blot, Generated, Inhibition, Staining, Fluorescence, Quantitation Assay, Software, Standard Deviation, SDS Page

Journal: Nucleic Acids Research

Article Title: DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

doi: 10.1093/nar/gkad549

Figure Lengend Snippet: Deacetylation by SIRT2 directs DNA-PK activation and signaling. ( A ) WT and SIRT2 KO U2OS cells were treated with or without 10 Gy IR. Cells were fixed at 30, 60 or 90 min post IR and stained with anti-γH2AX (red), anti-DNA-PKcs pS2056 (green) and DAPI (blue). A representative 90 min post IR time point is shown. ( B ) Quantitation of the percentage of cells with >5 DNA-PKcs pS2056 foci is shown for (A). The mean from each group was calculated from three biological replicates (100 cells per replicate) and error bars represent S.D. * P < 0.05; ** P < 0.01. ( C ) WT and SIRT2 KO U2OS cells were transfected with SIRT2-FLAG WT, H187Y or mock transfected and treated with or without 10 Gy IR. Cells were fixed at 30, 60 or 90 min post IR and stained with anti-FLAG (red), anti-DNA-PKcs pS2056 (green) and DAPI (blue). A representative 90 min post IR time point is shown. ( D ) Quantitation of the percentage of cells with >5 DNA-PKcs pS2056 foci is shown for (C) and Figure S3. The mean for each group was calculated from three biological replicates (100 cells per replicate) and error bars represent S.D. * P < 0.05; ** P < 0.01. ( E ) Western blot analysis for (C) and (D). ( F ) Western blot analysis of DNA-PKcs autophosphorylation at S2056 in response to IR in WT and SIRT2 KO HCT116 cells transfected with or without SIRT2-FLAG WT and H187Y. Quantification and statistical analysis is shown in . ( G ) Western blot analysis of Artemis and XRCC4 phosphorylation at S516 and S260 respectively in response to IR in WT and SIRT2 KO HCT116 cells transfected with or without SIRT2-FLAG WT and H187Y.

Article Snippet: Cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05–636), DNA-PKcs phosphoserine 2056 (Abcam, ab18192), anti-FLAG (Cell Signaling: 2368P), Artemis pS516 (Abcam, ab138411) or XRCC4 pS260 (Sigma, HPA006801) primary antibodies, followed by Alexa Fluor 555/488 secondary antibodies and DAPI.

Techniques: Activation Assay, Staining, Quantitation Assay, Transfection, Western Blot

Journal: Nucleic Acids Research

Article Title: DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

doi: 10.1093/nar/gkad549

Figure Lengend Snippet: Model of SIRT2 regulation of DNA-PKcs in NHEJ repair. DNA damage results in a DSB which leads to the recruitment of Ku70 and Ku80 heterodimers to both sides of the DSB . SIRT2 deacetylates DNA-PKcs in response to damage on its N terminus (light brown), which is important for DNA-PKcs localization to the sites of DNA damage, its interaction with Ku, and the formation of DNA-PK holoenzyme dimers . Once dimerized, DNA-PK phosphorylates downstream NHEJ repair factor Artemis, and Artemis resects DNA ends ( , ). DNA-PKcs autophosphorylates at serine 2056 and phosphorylates XRCC4 . XRCC4 with DNA ligase 4 and DNA-PKcs promotes end ligation . DNA-PKcs releases the repaired DNA .

Article Snippet: Cells were blocked in 5% BSA and immunostained with anti-γH2AX (Millipore 05–636), DNA-PKcs phosphoserine 2056 (Abcam, ab18192), anti-FLAG (Cell Signaling: 2368P), Artemis pS516 (Abcam, ab138411) or XRCC4 pS260 (Sigma, HPA006801) primary antibodies, followed by Alexa Fluor 555/488 secondary antibodies and DAPI.

Techniques: Ligation

Journal: International Journal of Molecular Sciences

Article Title: AMPK/FOXO3a Pathway Increases Activity and/or Expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 and Induces Radioresistance under Nutrient Starvation

doi: 10.3390/ijms241612828

Figure Lengend Snippet: Effects of nutrient starvation on clonogenic cell survival and phosphorylation and/or expression of AMPKα, ATM, DNA-PKcs, FOXO3a, Src, EGFR, PDK1, SOD2, and HIF-1α in MDA-MB-231 cells. ( A ) MDA-MB-231 cells were cultured in glucose-free DMEM without FBS for nutrient starvation (FBS(−) and Glc(−)) or control DMEM with 10% FBS (FBS(+) and Glc(+)) for 12 h before 0–6 Gy irradiation. After irradiation, the cells were cultured under the same condition for 4 h and used for a colony formation assay. The data are presented as the mean ± SD from three independent experiments (*: p < 0.05). ( B , C ) MDA-MB-231 cells were cultured under nutrient starvation for 0–24 h and processed for Western blot analyses with the antibodies indicated. Western blot results at the 0-h time point showed the expression of AMPKα, ATM, DNA-PKcs, FOXO3a, SOD2, Src, EGFR, PDK1, and HIF-1α when the cells were cultured in the control DMEM containing 1.0 g/L glucose with 10% FBS.

Article Snippet: The following antibodies were used as primary antibodies: AMPKα antibody (#2603, Cell Signaling Technology, Inc. (CST), Beverly, MA, USA); phospho-AMPKα (Thr172) antibody (#2535, CST); ATM antibody (#NB100-104, Novus Biologicals, LLC, Englewood, CO, USA); phospho-ATM (Ser1981) antibody (#5883, CST); DNA-PKcs antibody (#sc-9051, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); phospho-DNA-PKcs (Ser2056) antibody (#ab18192, Abcam, PLC, Cambridge, UK); EGFR antibody (#2232, CST); phospho-EGFR (Tyr845) antibody (#2231, CST); FOXO3a antibody (#12829, CST); phospho-FOXO3a (Ser413) antibody (#8174, CST); hypoxia-inducible factor-1 alpha (HIF-1α) antibody (#A300-286A, Bethyl Laboratories, Inc., Montgomery, TX, USA); PDK1 antibody (#3062, CST); phospho-PDK (Ser241) antibody (#3438, CST); SOD2 antibody (#13141, CST); Src antibody (#2108, CST); and phospho-Src (#2102, CST) antibody.

Techniques: Expressing, Cell Culture, Irradiation, Colony Assay, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: AMPK/FOXO3a Pathway Increases Activity and/or Expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 and Induces Radioresistance under Nutrient Starvation

doi: 10.3390/ijms241612828

Figure Lengend Snippet: Effects of AMPKα knockdown on phosphorylation and/or expression of ATM, DNA-PKcs, FOXO3a, Src, EGFR, PDK1, and SOD2 in MDA-MB-231 cells. ( A , B ) MDA-MB-231 cells treated with siRNA for AMPKα (siAMPKα) or with control siRNA (siCtrl) were cultured in glucose-free DMEM without FBS for nutrient starvation (FBS(−) and Glc(−)) or in control DMEM with 10% FBS (FBS(+) and Glc(+)) for 12 h and processed for Western blot analyses with the antibodies indicated.

Article Snippet: The following antibodies were used as primary antibodies: AMPKα antibody (#2603, Cell Signaling Technology, Inc. (CST), Beverly, MA, USA); phospho-AMPKα (Thr172) antibody (#2535, CST); ATM antibody (#NB100-104, Novus Biologicals, LLC, Englewood, CO, USA); phospho-ATM (Ser1981) antibody (#5883, CST); DNA-PKcs antibody (#sc-9051, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); phospho-DNA-PKcs (Ser2056) antibody (#ab18192, Abcam, PLC, Cambridge, UK); EGFR antibody (#2232, CST); phospho-EGFR (Tyr845) antibody (#2231, CST); FOXO3a antibody (#12829, CST); phospho-FOXO3a (Ser413) antibody (#8174, CST); hypoxia-inducible factor-1 alpha (HIF-1α) antibody (#A300-286A, Bethyl Laboratories, Inc., Montgomery, TX, USA); PDK1 antibody (#3062, CST); phospho-PDK (Ser241) antibody (#3438, CST); SOD2 antibody (#13141, CST); Src antibody (#2108, CST); and phospho-Src (#2102, CST) antibody.

Techniques: Expressing, Cell Culture, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: AMPK/FOXO3a Pathway Increases Activity and/or Expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 and Induces Radioresistance under Nutrient Starvation

doi: 10.3390/ijms241612828

Figure Lengend Snippet: Effects of FOXO3a knockdown on phosphorylation and/or expression of AMPKα, ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 in MDA-MB-231 cells. ( A , B ) MDA-MB-231 cells treated with siRNA for FOXO3a (siFOXO3a) or with control siRNA (siCtrl) were cultured in glucose-free DMEM without FBS for nutrient starvation (FBS(−) and Glc(−)) or in control DMEM with 10% FBS (FBS(+) and Glc(+)) for 12 h and processed for Western blot analyses with antibodies indicated.

Article Snippet: The following antibodies were used as primary antibodies: AMPKα antibody (#2603, Cell Signaling Technology, Inc. (CST), Beverly, MA, USA); phospho-AMPKα (Thr172) antibody (#2535, CST); ATM antibody (#NB100-104, Novus Biologicals, LLC, Englewood, CO, USA); phospho-ATM (Ser1981) antibody (#5883, CST); DNA-PKcs antibody (#sc-9051, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); phospho-DNA-PKcs (Ser2056) antibody (#ab18192, Abcam, PLC, Cambridge, UK); EGFR antibody (#2232, CST); phospho-EGFR (Tyr845) antibody (#2231, CST); FOXO3a antibody (#12829, CST); phospho-FOXO3a (Ser413) antibody (#8174, CST); hypoxia-inducible factor-1 alpha (HIF-1α) antibody (#A300-286A, Bethyl Laboratories, Inc., Montgomery, TX, USA); PDK1 antibody (#3062, CST); phospho-PDK (Ser241) antibody (#3438, CST); SOD2 antibody (#13141, CST); Src antibody (#2108, CST); and phospho-Src (#2102, CST) antibody.

Techniques: Expressing, Cell Culture, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: AMPK/FOXO3a Pathway Increases Activity and/or Expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 and Induces Radioresistance under Nutrient Starvation

doi: 10.3390/ijms241612828

Figure Lengend Snippet: Suggested molecular pathway for the increased activity and/or expression of AMPK, FOXO3a, ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2 under nutrient starvation. Our results suggest that nutrient starvation activates the AMPK/FOXO3a pathway and induces radioresistance via increased activity and/or expression of ATM, DNA-PKcs, Src, EGFR, PDK1, and SOD2. AMPK and FOXO3a appear to be key molecules that induce radioresistance under nutrient starvation. Purple arrows indicate activation pathways presented in this manuscript. Red arrows indicate transcriptional activation presented in this manuscript. Black arrows indicate previously reported pathways. Blue arrow indicates transcription.

Article Snippet: The following antibodies were used as primary antibodies: AMPKα antibody (#2603, Cell Signaling Technology, Inc. (CST), Beverly, MA, USA); phospho-AMPKα (Thr172) antibody (#2535, CST); ATM antibody (#NB100-104, Novus Biologicals, LLC, Englewood, CO, USA); phospho-ATM (Ser1981) antibody (#5883, CST); DNA-PKcs antibody (#sc-9051, Santa Cruz Biotechnology, Inc., Dallas, TX, USA); phospho-DNA-PKcs (Ser2056) antibody (#ab18192, Abcam, PLC, Cambridge, UK); EGFR antibody (#2232, CST); phospho-EGFR (Tyr845) antibody (#2231, CST); FOXO3a antibody (#12829, CST); phospho-FOXO3a (Ser413) antibody (#8174, CST); hypoxia-inducible factor-1 alpha (HIF-1α) antibody (#A300-286A, Bethyl Laboratories, Inc., Montgomery, TX, USA); PDK1 antibody (#3062, CST); phospho-PDK (Ser241) antibody (#3438, CST); SOD2 antibody (#13141, CST); Src antibody (#2108, CST); and phospho-Src (#2102, CST) antibody.

Techniques: Activity Assay, Expressing, Activation Assay