EMBO/SourceData huggingface dataset:
Figure 5(A) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.(C) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.(G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.
Figure 1(A) THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.(B) THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS HSV-1 and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(C) THP1 cells were treated with infectious or UV-inactivated HSV-1 (KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) MDMs were infected with ICP0-deficient or revertant HSV-1 (KOS), MOI 3. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(E) THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 µg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.
Figure 2(A) MDMs were infected for 18 h with HSV-1 (KOS), MOI 1. Cytosolic viral proteins were detected by mass spectrometry using iTRAQ-labeling. Identified viral proteins are shown.(B) MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting.(C) HFFs and MDMs were infected with HSV-1 (KOS) MOI 0.1 and 1.0. Culture medium was replaced 3 hpi, and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates +/- st.dev.(D) THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 hrs. Cell lysates were isolated and levels of ICP27 and β-actin were determined by Western blotting.
Figure 3(A) THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 hours for measurement of type I IFN bioactivity.(B) MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(C) THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2x104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post transfection.(E) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity
Figure 4(A) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.(B) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post infection, and stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar 10 μm.(C, D) THP1 cells were infected with HSV-1 KOS, ΔICP27 or HSV-1 ΔNLS. The localization of the major NLS is illustrated in panel C. Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(E, F) THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post infection. Western blotting was used to determine levels of phospho- and total TBK1 were determined in the cytoplasmic fractions and levels of phospho-IRF3 and RCC1 in the nuclear fractions.(G-I) HEK293T cells were (G) transfected with STING, TBK1, IRF3-5D, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.(G-I) HEK293T cells were ((H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.(G-I) HEK293T cells were (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post transfection.
Figure 5(A) Lysates from THP1 cells infected with HSV-1 KOS (MOI 10) were subjected to immunoprecipitation using an anti-STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting.(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-pTBK1 (B). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and pTBK1. Scale bar 10 μm.(B, C) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti-ICP27 together with anti-STING (C). The stainings were visualized by confocal microscopy. Arrowheads, colocalizations between ICP27 and STING. Scale bar 10 μm.(D) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING.(E) THP1 control and STING KO cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27, phospho-TBK1 (pTBK1), and TBK1.(F) THP1 control and TBK1 KO cells were infected with HSV-1 KOS (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.(G) THP1 cells were infected with HSV-1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.(H) THP1 cells were infected with HSV-1 KOS (MOI 10) for 6 and 8 h and total lysates were generated. Phospho-TBK1 was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and total TBK1.(I) TBK1 was immunoprecipitated from whole cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.
Figure 6(C) THP1 cells were infected with HSV-1 (KOS), HSV-2 (HG52), HSV-1 ΔICP27, or HSV-1 ΔICP27 rescued with HSV-2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(D) RAW264.7 cells were infected with HSV-1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.(F) THP1 cells were infected with HSV-1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting.(G) THP1 cells were infected with HSV-1 KOS or HSV-1 ΔRGG (MOI 10) for 8 h and total lysates were generated. STING was immunoprecipitated, and subjected to Western blotting using antibodies against ICP27 and STING.
Figure 1A. Results of CRISPR screening analyzed by the MAGeCK algorithm.B. Wild type (WT) and NCOA4 KO MEFs were pretreated with 25 μg/ml ferric ammonium citrate (FAC) for 16 h and then chased with 20 μg/ml cycloheximide (CHX) for the indicated times. Soluble ferritin expression levels were determined by immunoblotting with the indicated antibodies.C. WT MEFs untreated or treated with 10 μg/ml FAC for 12 h were lysed with SDS-containing sample buffer or Triton buffer, and analyzed by immunoblotting with the indicated antibodies.E. Subcellular fractions of MEFs treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12 h, or 100 μM Deferoxamine (Dfo) for 6 h after treatment with 10 μg/ml FAC for 12 h were prepared by differential centrifugation as described in (D). Each fraction was subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.F. WT MEFs were untreated, treated with 10 μg/ml FAC for 12h or treated with 10 μg/ml FAC, 10 μM E64d and 10 μg/ml pepstatin A (pep A) for 12h. The cells were immunostained with anti-NCOA4 and anti-LAMP1. Scale bar, 10 μm. Quantitative data are shown as the means ± SD of three biological replicates. At least 30 cells were quantified in each replicate. P=0.033 (Welch Two Sample t-test).G. NCOA4 puncta were photobleached in MEFs stably expressing NCOA4-GFP after treatment with 10 μg/ml FAC for 12 h, and then fluorescent recovery was monitored. p62 puncta were photobleached in MEFs stably expressing GFP-human p62, and then fluorescent recovery was monitored. Representative images are shown. Time 0 indicates the start of recovery after photobleaching. Scale bars, 2 μm. H. Quantitative data of fluorescence recovery in (G) shown as means ± SD. 26 dots (NCOA4) or 25 dots (p62) were quantified from three biological replicates.I. Time-lapse imaging of MEFs stably expressing NCOA4-GFP. Representative fusion and fission images are shown. Red arrowheads indicate the fission event. Scale bars, 5 μm.
Figure 2B. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h and then fixed for imaging. Cells were immunostained with anti-myc antibody. Representative images are shown. Scale bar, 10 μm.C. Number of puncta per cell in (B) are shown as the means ± SEM of at least 40 cells in each condition from two biological replicates.D. NCOA4 KO MEFs reconstituted with NCOA4 variants were treated or not treated with 10 μg/ml FAC for 12 h, fractionated, and analyzed by immunoblotting with the indicated antibodies.E. DISOPRED3 disorder score of human NCOA4.F. WT MEFs stably expressing NCOA4 IDR (aa 167-334) fused with tandem FKBP were treated with 10 μg/ml FAC or 20 μM Dfo for 12 h and then cultured with 0.1 nM FKBP ligand (AP20187) for 6h. The cells were fixed for imaging and immunostained with anti-myc to analyze puncta of the fusion protein. Scale bar, 10μm. G. Number of puncta in (F) are shown as means ± SEM of at least 35 cells in each condition from two biological replicates. P<0.0001 (Dfo+ligand vs FAC+ligand) and P=0.048 (FAC vs FAC+ligand) (Kruskal-Wallis ANOVA with Dunn's multiple comparison test). P values were adjusted using the Bonferroni method.H. Coomassie-stained SDS-PAGE gel of purified proteins. I. The amount of co-purifying iron in purified proteins in (H) was measured by inductively coupled plasma mass spectrometry (ICP-MS). Data are shown as means ± SD of three biological replicates. P=0.032 (Welch Two Sample t-test).
Figure 3A. Purified NCOA4 proteins were incubated with FeSO4 for 1 h under aerobic conditions and fractionated. Samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B. Quantitative results in (A) are shown as means ± SEM of three biological replicates.F. Electron micrograph of NCOA4 KO MEFs reconstituted with myc-hNCOA4. Cryosections were labeled with anti-myc antibody. Scale bar, 200nm. G. Quantification of the diameter of NCOA4 condensates. Red line shows the mean. The dot plot represents the diameter of 36 condensates from two biological replicates.
Figure 4A. MEFs were treated with 10 μg/ml FAC for the indicated times and harvested. Cell lysates were fractionated and analyzed by SDS-PAGE with the indicated antibodies. Asterisks indicate a non-specific band. TfR1; Transferrin receptor 1B. MEFs were cultured for 12 h in medium containing 10 μg/ml FAC supplemented with DMSO or 10 μM E64d and 10 μg/ml pepstatin A (pepA). Samples were analyzed by immunoblotting with the indicated antibodies. The black arrowhead indicates an intact ferritin band, and the white arrowhead indicates a partial degradation band of ferritin.C. NCOA4 KO MEFs reconstituted with hNCOA4-myc were cultured with 10 μg/ml FAC for the indicated times and fixed for imaging. Cells were immunostained with ferritin and myc antibodies. Scale bar, 10μm.D. Myc (NCOA4) puncta were extracted, and average signal intensities of myc and ferritin in the puncta in (C) were plotted.E. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 6 h or 24 h and then cultured with DMSO or 50 μM biotin for 30 min. Cells were lysed with Triton buffer, and lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed with immunoblotting by indicated antibodies.F. WT MEFs pretreated with 10 μg/ml FAC for 6 h or 24 h were chased with 20 μg/ml CHX and analyzed by immunoblotting with the indicated antibodies.G. NCOA4 KO MEFs reconstituted with myc-hNCOA4 were pretreated with 10 μg/ml FAC and then cultured with 20 μg/ml CHX or 200 nM bafilomycin A1 (BafA). Cells were fixed for imaging and immunostained using myc and LAMP1 antibodies. Scale bar, 10μm.
Figure 5A. WT, ATG7 KO, TAX1BP1 KO, and FIP200KO MEFs were treated or not treated with 10 μg/ml FAC for 12 h. Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.C. HEK293T cells were transfected with GFP-NCOA4 and the indicated FLAG-His-TAX1BP1 variants. Soluble lysates extracted from transfected cells were immunoprecipitated with anti-FLAG antibody. Inputs and immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were untreated or treated with 10 μg/ml FAC for 12 h. (D, F) Cell lysates were fractionated and analyzed by immunoblotting with the indicated antibodies.WT MEFs and TAX1BP1 KO MEFs reconstituted with the indicated TAX1BP1 variants were treated with 10 μg/ml FAC for 12 h. (E) Cells were fixed for imaging and immunostained to analyze ferritin localization. Scale bar, 10μm.
Figure 6A. MEFs were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo for the indicated time. Cells were lysed with SDS-containing sample buffer or Triton buffer. Lysates were analyzed by immunoblotting with the indicated antibodies.B. MEFs were treated with 20 μM Dfo for 12 h, 10 μg/ml FAC for 12h, or 100 μM Dfo for 6 h after treatment with 10 μg/ml FAC for 12 h. Triton-soluble lysates were subjected to glycerol gradient ultracentrifugation analysis.C. WT and TAX1BP1 KO MEFs were pretreated with 25 μg/ml FAC for 16 h and then cultured with 100 μM Dfo. Lysates were fractionated and then analyzed by immunoblotting with the indicated antibodies. D. Soluble FTL bands in (C) were quantified by densitometry. Data are shown as the means ± SD of three biological replicates.E. TAX1BP1 KO MEFs (sg1 clone) stably expressing FLAG-OsTIR1 (F74G) and FLAG-mAID-mTAX1BP1 were pretreated with 10 μg/ml FAC for 12 h, cultured with DMSO or 1 μM 5-Ph-IAA for 2 h, and then treated with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies. 5-Ph-IAA, a derivative of Auxin; mAID, mini auxin-inducible degronF. TAX1BP1 KO MEFs (clone sg1) in which ATG7 was knocked down were pretreated with 10 μg/ml FAC for 12 h and then cultured with 100 μM Dfo. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.G. TAX1BP1 KO MEFs (clone sg1) stably expressing myc-hNCOA4 pretreated with 25 μg/ml FAC for 16 h were cultured with 100 μM Dfo, and soluble lysates were analyzed by immunoblotting with the indicated antibodies.H, I. NCOA4 KO MEFs reconstituted with NCOA4 variants were pretreated with 10 μg/ml FAC for 12 h and then chased with (H) 20 μg/ml CHX or (I) 100 μM Dfo for the indicated times. Soluble lysates were analyzed by immunoblotting with the indicated antibodies.J. MEFs expressing hNCOA4-TurboID were treated with 10 μg/ml FAC for 3 h (indicated as F) or 100 μM DFO for 3 h after treatment with 10 μg/ml FAC for 12 h (indicated as D), and then cultured with DMSO or 50 μM biotin for 30 min. Triton-soluble cell lysates were pulled down by streptavidin beads. Inputs and pulldown samples were analyzed by immunoblotting with the indicated antibodies. The black and white arrowheads indicate hNCOA4-TurboID and endogenous NCOA4, respectively.
Figure 1A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.
Figure 2B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.
Figure 3A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05