Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 3. Impaired Tip60 expression in RA patients. CD4+CD45RO−T cells from RA patients and controls were stimulated for 4 days, and (A–B) Tip60 gene expression was quantified by QPCR in naïve T cells from RA patients, HCs and OA patients. The results from 3 independent experiments involving 16 RA patients and 14 HCs are shown as scatter plots in relation to the donor age (A) and as bar graphs displaying the mean ± SEM of the RA patients, HCs and OA patients (B). (C) Tip60 insufficiency is independent of drug treatment (treated vs. treatment naïve), and Tip60 gene expression was measured in the naïve CD4+ T cells 4 days after TCR-mediated stimulation. (D) Tip60 transcript levels were correlated with the number of Treg cells in the PBMCs from RA patients. (E–F) Representative blotting for Tip60 and Foxp3 protein expression (E). The Tip60 and Foxp3 band intensities were adjusted to GAPDH. The western blotting experiment results analyzing 6 RA patients, 6 OA and 6 HCs are shown as the mean ± SEM (F). (G) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue sections from the OA and RA patients. Bar, 20 μm. (H) Fluorescence intensities of Foxp3 (green), acetylated-lysine (red), acetylated-Foxp3 (combined green and red), and Tip60 (white) in the sections of synovial tissue from 6 OA and 6 RA patients are presented as the mean ± SEM. ***P < 0.001; n.s., non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Gene Expression, Western Blot, Immunostaining, Fluorescence

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 4. Foxp3 expression and Treg cell differentiation are regulated by Tip60. CD4+ T cells from healthy donors were transfected with control or Tip60-specific shRNA by nucleofection or treated with Tip60-specific inhibitor NU9056 (2 μM) at day 3 after bead stimulation. (A) The Tip60 transcript levels were quantified by QPCR 24 h after control and Tip60 siRNA transfection. (B) Representative blotting for Tip60 and Foxp3 expression. (C) Foxp3 acetylation was determined by a coimmunoprecipitation assay after transfection. (D) The Foxp3 levels in T cells were analyzed by flow cytometry in the GFP+ cell population after control and Tip60 shRNA transfection. Representative histograms for one control shRNA (blue) and one Tip60 shRNA (red); three independent experiments examining 6 HCs are presented as the mean ± SEM. (E–G) CD4+ T cells were transfected with the control or Tip60 siRNA and cultured under Treg- or Th17-polarizing conditions, and intracellular staining for Foxp3 and IL-17a was performed. (E) Representative dot plots for Treg and Th17 cell production. (F) Percentages of Tregs under Treg- polarizing conditions or CD4+Foxp3+ cells and CD4+IL-17a+ Th17 cells under Th17-polarizing conditions are presented as the mean ± SEM. (G) Suppression ability of the Tregs from control and Tip60 siRNA was analyzed by quantifying CFSE dilution. (H–M) Tip60 inhibitor: (H) Representative blotting for Foxp3 protein expression after NU9056 treatment. (I) Foxp3 acetylation was determined by a coimmunoprecipitation assay. (J) The Foxp3 protein levels in T cells were analyzed by flow cytometry. Representative histograms for DMSO (blue) and the Tip60 inhibitor (red); three independent experiments examining 6 HCs are presented as the mean ± SEM. (K–M) CD4+ T cells were treated with vehicle (DMSO) or NU9056 (2 μM) and cultured under Treg- or Th17-polarizing conditions. (K) Representative dot plots. (L) Percentages of Tregs or Th17 cells are presented as the mean ± SEM. (M) Suppression ability of the Tregs from vehicle and inhibitor treatment cells was analyzed by quantifying CFSE dilution. **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Transfection, Control, shRNA, Co-Immunoprecipitation Assay, Cytometry, Cell Culture, Staining

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 5. Overexpression of Tip60 restores Treg cell differentiation. Naïve CD4+ T cells from RA patients were transduced with pEGFP-C2 or pEGFP-Tip60. The cells were transfected after anti-CD3/CD28 bead stimulation. (A) Representative blotting for Tip60 and Foxp3 protein expression after transfection. (B–C) The Foxp3 protein levels in naïve T cells were analyzed by flow cytometry in GFP+ cell population after transfection (B). Three independent experiments examining 6 RA patients are presented as the mean ± SEM (C). (D) Foxp3 acetylation was determined by a coimmunoprecipitation assay with anti-Foxp3, followed by im- munoblotting with an anti-acetylated-lysine antibody. Representative blots from three independent experiments are shown. (E–H) T cell differentiation: CD4+CD45RO−T cells were cultured under Treg- or Th17-polarizing conditions, re-stimulated with PMA/ionomycin and subjected to intracellular staining for Foxp3 and IL-17a. (E–F) Representative dot plots for Tregs (right) and Th17 cell (left) production. (G) Percentages of Tregs under Treg-polarizing condition or CD4+Foxp3+ cells and CD4+IL-17a+ Th17 cells under Th17-polarizing conditions from three independent experiments examining 6 RA patients are presented as the mean ± SEM. (H) Naïve CD4+ T cells from RA patients were transduced with pcDNA3.1and pcDNA3.1-Tip60. Suppression capability of the Tregs from cells transfected with control and Tip60 plasmids was analyzed by quantifying the CFSE dilution. The mean ± SEM of division indics are shown from 6 independent coculture experiments. **P < 0.01; ***P < 0.001.

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Over Expression, Transduction, Transfection, Expressing, Cytometry, Co-Immunoprecipitation Assay, Cell Culture, Staining, Control

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 6. Tip60 controls the arthritogenic effect of T cells. Pairs of NSG mice were engrafted with synovial tissue from RA patients, CD45RO−PBMCs from healthy donors were transferred to the chimeric mice. The mice were divided into two groups, vehicle (DMSO) control and treatment with NU9056 group, where both treatments were delivered by intraperitoneal injection of 2 mg/kg/day for 9 days. (A) H&E staining was performed to compare the overall morphology of the synovium. Representative images of the synovium in the vehicle (n = 6) and Tip60 inhibitor-treated (n = 6) chimeric mice are shown. The data are representative of three independent experiments. (B) The intensity of synovial inflammation was compared by QPCR to assess TRB and TNFSF11 gene expression. All data are presented as the mean ± SEM from at least 6 different synovial grafts. (C–E) CD20 (C) and RANKL (D) were stained by immunohistochemistry in synovial tissue sections. The percentages of CD20+ B cells and RANKL-positive cells were determined by counting 9 different fields of synovial tissue sections and presented as the mean ± SEM (E). (F–H) T cell infiltration: (F) Representative images of anti-CD3 (red) and IL-17a staining (green) of the vehicle- (n = 6) and Tip60 inhibitor-treated (n = 6) chimeric mice. CD3+IL-17+ double positive cells are indicated by a yellow coloring. Bar, 20 μm. (G) Representative images of anti-Foxp3 staining (green) of the vehicle- (n = 6) and Tip60 inhibitor-treated (n = 6) samples. Bar, 20 μm. (H) The percentages of CD3+IL-17+ cells and Foxp3+ cells are presented as the mean ± SEM. (I) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue from vehicle and Tip60 inhibitor-treated chimeric mice. Bar, 20 μm. (J) Fluorescence intensity of Foxp3, acetylated-Foxp3 (combined green and red) and Tip60 are presented as the mean ± SEM. (K) Expression of IL-6, TNF-α, IL-17, IL-10 and TGF-β in the synovium was measured by QPCR. The data are representative of three independent experiments. ***P < 0.001; n.s., non-significance. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Control, Injection, Staining, Gene Expression, Immunohistochemistry, Immunostaining, Fluorescence, Expressing

Journal: Journal of autoimmunity

Article Title: Impaired Tip60-mediated Foxp3 acetylation attenuates regulatory T cell development in rheumatoid arthritis.

doi: 10.1016/j.jaut.2019.02.007

Figure Lengend Snippet: Fig. 7. Restoring Tip60 expression in RA T cells prevents arthritogenic effect. Pairs of NSG mice were engrafted with synovial tissue from RA patients and divided into two treatment groups. CD45RO−PBMCs from RA patients were transfected with either a control plasmid (pcDNA3.1) or pcDNA3.1-Tip60 and adop- tively transferred to the chimeric mice. (A) H&E staining to compare the overall morphology of the synovium from control and Tip60 plasmids transferred chimeric mice. Representative images of the synovium in the control (n = 6) and Tip60 plasmids (n = 6) samples are shown. Bar, 20 μm. (B) The intensity of synovial inflammation was compared by QPCR to assess TRB and TNFSF11 gene expression. The data are representative of three independent experiments. All data are presented as the mean ± SEM from at least 6 different synovial grafts. (C–E) CD20 (C) and RANKL (D) were stained by immunohistochemistry in synovial tissue sections from control and Tip60 plasmid-transfected chimeric mice. The percentages of CD20+ B cells and RANKL-positive cells are presented as the mean ± SEM (E). (F–H) T cell infiltration: (F) Representative images of anti-CD3 (red) and IL-17 staining (green) of the control (n = 6) and Tip60 plasmids (n = 6) samples. CD3+IL-17a+ cells are indicated with a yellow coloring. Bar, 20 μm. (G) Representative images of anti-Foxp3 staining (green) of the control (n = 6) and Tip60 plasmids (n = 6) samples. Bar, 20 μm. (H) The percentages of CD3+IL-17a+ cells and Foxp3+cells in randomly selected fields of synovial tissue sections are presented as the mean ± SEM. (I) Co-immunostaining of Foxp3 (green), acetylated-lysine (red), and Tip60 (white). Representative images are from one of three synovial tissue from control and Tip60 vector transfected mice. Bar, 20 μm. (J) Fluorescence intensity of Foxp3, acetylated-Foxp3 (combined green and red) and Tip60 are presented as the mean ± SEM. (K) Expression of IL-6, TNF-α, IL-17, IL-10 and TGF-β in the synovium was measured by QPCR. The data are representative of three independent experiments. **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Alternatively, Oligo duplex RNA specific for Tip60 and a control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Staining, Gene Expression, Immunohistochemistry, Immunostaining, Fluorescence

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 1 PHF8 and TIP60 colocalize and recruit each other in neuronal interchromatin space. A, Endogenous PHF8 immunostained with anti-PHF8 antibody (ab36068; Abcam) forms hundreds of discrete puncta that specifically localize to the interchromatin space in hippocampal neurons (representative z-slice of a hippocampal neuronal nucleus; left, widefield; right, SIM). Scale bar, 1 m. B, Endogenous TIP60 forms puncta of roughly the same caliber as those of PHF8 above (left, hippocampal nucleus in widefield; right, SIM), which also localize to the interchromatin space. Scale bar, 1 m. C, A representative field of hippocampal neurons stained with an antibody against the phosphorylated CTD of RNA polymerase II (YSPTSPS phospho S5, abbreviated to S5P), showing that S5P, a marker of the transcription initiation complex, localizes to the same nuclear compartment as PHF8 in the nucleus. Scale bar, 1 m. D, A hippocampal neuronal nucleus outlined in blue, showing the localization of spectrally distinct PHF8tdTomato and TIP60CFP pseudo-colored in green and red, respectively, which overlapped completely in the nuclear interchromatin space (merge channel, yellow pixels indicate colocalization). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. E, When TIP60 is overexpressed by itself in hippocampal neurons (middle, red), endogenous PHF8 (left, green) is seen to be recruited to the TIP60 puncta in hippocampal neurons (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. Asterisk () indicates endogenous protein staining. F, When PHF8 is overexpressed by itself (left, green) in hippocampal neurons, endogenous TIP60 (middle) is seen to be recruited to the PHF8 puncta (right, merge). DAPI was used to stain the DNA (blue). Scale bar, 0.2 m. Asterisk () indicates endogenous protein staining. G. Endogenous TIP60 is located within 30 nm of PHF8 as shown by P-LISA, showing distinct areas where PHF8 interacts with endogenous TIP60 (red spots) on the border with DAPI-dense regions (blue). Scale bar, 0.5 m. H, Two Hek293 nuclei are shown, one is positive for PHF8 shRNA (outlined in red) while the other is not (outlined in green). Positive PHF8TIP60 interaction hotspots were stained as red punctae. Scale bar, 0.5 m. I, Quantification of the number of hotspots found in cells transfected with PHF8, PHF8 shRNA, the mutant PHF8 F279S, or, in F279S-transfected cells expressing PHF8, shRNA were quantified using Blobfinder, and the means and standard errors are displayed in a bar grapht (triple asterisks indicating statistical significance using the unpaired t test; p 0.0001). J, Double immunofluorescence confirming the existence of PHF8 in the identified PLA hotspots where PHF8 and TIP60 interact. Scale bar, 0.5 m.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Staining, Marker, shRNA, Transfection, Mutagenesis, Expressing, Immunofluorescence

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 2 PHF8 and TIP60 physically associate to form a dual function chromatin-modifying complex. A, Coimmunoprecipitation of PHF8 and TIP60 in HEK293T nuclear extracts, where TIP60YFP was pulled down with anti-GFP antibody and PHF8FLAG was detected with anti-FLAG by Western blotting. B, Pulldown of PHF8YFP showed that TIP60FLAG was detected in the IP fraction but not in the YFP-only control lane. C, Endogenous coimmunoprecipitation of PHF8 and TIP60 in DIV12 cortical neuronal nuclear extracts, showing that PHF8 is able to be pulled down by both the anti-PHF8 antibody and anti-TIP60 antibody, but not the anti-GFP antibody. D, E, Truncated constructs of TIP60 protein (A to F) containing the indicated TIP60 domain (E) were fused to YFP and then cotransfected with full-length PHF8FLAG and immunoprecipitated with an anti-GFP antibody. Western analysis was performed to detect PHF8FLAG in the immunoprecipitates using the anti-FLAG antibody. A negative control of YFP only is denoted by (-), whereas full-length TIP60 served as a positive control (). F, G, Total histones from HEK293 cells overexpressing PHF8, TIP60, or both were separated on TAU gels (F) or conventional SDS-PAGE (G). Overexpression of TIP60 alone increases H3K9 acetylation in HEK293 cells for both the H3.1 and H3.3 isoforms, whereas acetylation of the non-TIP60 substrate H2BK5 was not affected. Coexpression of PHF8 and TIP60 increases H3.3K9 acetylation to even higher levels. H, Chromatin immunoprecipitation using an antibody specific to H3K9me2, showing that overexpression of wild-type PHF8 but not the clinical mutant F279S (U, unbound or input levels of H3K9me2; B, bound or immunoprecipitated H3K9me2). I, J, ChIP assays of HEK293T cells transfected with PHF8, TIP60, or both analyzing histone tails positive for H3K4me3, the transcriptionally-activating histone mark that is known to be bound by PHF8, show that the increase in H3K9ac (I) and H3K14ac (J) is specific to histones carrying H3K4me3, and that this histone population was enriched in H3.3 (as shown by the more intense staining of this isoform on the TAU gel; asterisk). Western blot of the same lysates using an H3.3 antibody serves as loading control. The right panel shows bar graphs quantifying the increase in H3.3K9 and H3.3K14 acetylation, relative to the untransfected control (n 3; p 0.19 for PHF8 only, 0.04 for TIP60 only, 0.02 for PHF8TIP60; asterisks indicates statistical significance: p 0.05).

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Western Blot, Control, Construct, Immunoprecipitation, Negative Control, Positive Control, SDS Page, Over Expression, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Staining

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 4 Neuronal activity reorganizes PHF8 and TIP60 in the nucleus and effectuate histone methylation and acetylation changes. A, A representative image of a pair of hippocampal neuronal nuclei during the first 5 min of 4APBicFors treatment and then at 45 min, showing the activity-dependent increase of PHF8 and TIP60 protein in the nucleus. B, Neural network activity visualized by Ca2

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activity Assay, Methylation

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 5 PHF8 and TIP60 modulate neuronal activity-induced histone acetylation at H3K9acS10P and activation of the Arc gene. A, Representative microscopic field of hippocampal neurons after 1 h of network activation by chemLTP, showing a positive correlation between the expression of Arc (red) and Tip60 (blue) with the phosphoacetylation mark H3K9acS10P (green). The bottom panels show three different neurons that induced varying amounts of ARC protein. The neuron expressing the highest amount of ARC (3) also has high amounts of H3K9acS10P. B, Quantification of 20 immunofluorescence-analyzed fields exemplified in A, showing a statistically significant increase in H3K9acS10P as well as endogenous TIP60 in ARC-expressing neurons (n 347 neurons; p 0.00001). C, Fusion constructs of PHF8 and its mutant F279S were individually expressed in hippocampal neurons and the next day the neuronal network was activated using ChemLTP (4APBicFors). After 1 h of upregulated synaptic activity, the expression of PHF8, but not its mutant F279S, significantly increases histone acetylation at H3K9acS10P (n 397 neurons; p 0.00001). D, A representative microscopic field of neuronal nuclei after 1 h of ChemLTP, with neuronal nuclei stained by DAPI outlined in magenta,

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activity Assay, Activation Assay, Expressing, Immunofluorescence, Construct, Mutagenesis, Staining

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 7 PHF8 and TIP60 are actively recruited to specific neuronal gene promoters. A, B, Within minutes of synaptic activation (x-axis: time of increased network activity, in minutes), time-course ChIP shows an early detectable decrease in the chromatin mark H3K9me2 at the Arc TSS (A), which is mirrored by a concomitant, highly transient increase in the levels of H3K9acS10P at the Arc TSS (B). This increase in H3K9acS10P was specific to the Arc promoter as analyses of Rpl19, JunB, Synaptophysin, Homer1A, BDNF promoter 1, Txnip, Gapdh, and Fos intergenic region (Negative Ctrl) did not show an activity-dependent increase. C, D, Time-course ChIP followed by qRT-PCR using primers against the transcriptional start site regions of the Arc gene, arc synaptic response element, ribosomal protein L19 (Rpl19), neuronal PAS domain protein 4 (Npas4), and synaptophysin. Both TIP60 (C) and PHF8 (D) are recruited to the Arc TSS within minutes of activation of the neuronal network, but not to the Rpl19, Npas4, or Synaptophysin transcriptional start sites.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Activation Assay, Activity Assay, Quantitative RT-PCR

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 8 Common interacting partners between PHF8 and TIP60 function primarily in transcription and mRNA processing. Top, A Venn diagram showing several interacting partners of PHF8 and TIP60 as identified by immunoprecipitation followed by mass spectrometry. The overlapped region in the middle represents common partners that interact with PHF8 and TIP60, which include the splicing factor SFPQ (PSF) and its partner NONO, as well as several ATP-dependent RNA helicases, and the histone chaperone nucleolin. Proteins that have known acetylation sites are marked by a triangle (Choudhary et al., 2009). Arrows indicate known functional interactions between identified proteins. Font size indicates the percentage of the total protein that the identified MS/MS peptides covered (large font: 25% coverage; medium font: 5 25% coverage; small font: 5% coverage). Histone proteins identified in the IPMS are in bold. Bottom, A listing of the top eight biological functions attributed to the proteins identified in the IPMS of both PHF8 and TIP60 in order of abundance, as computed by the software DAVID (http://david.abcc.ncifcrf.gov/home.jsp), with the associated p value and Benjamini factor, showing that interactors of PHF8 and TIP60 are enriched in the functions of RNA processing, RNA splicing, and mRNA processing.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Immunoprecipitation, Mass Spectrometry, Functional Assay, Tandem Mass Spectroscopy, Software

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 9 Endogenous TIP60 is located within 30 nm of PHF8 in the activated hippocampal neuronal nucleus. A, A maximum intensity projection of a dual color 3D STORM image of a hippocampal neuronal nucleus that has undergone 1 h of chemLTP. The neuron has been labeled for endogenous TIP60 (red) and endogenous PHF8 (green), showing that the two molecules closely interact in various localized puncta in the nucleus. Scale bar, 1 m. The insets on the right show six representative complexes at higher magnification (scale bar, 50 nm). B, A highly magnified view of two endogenous PHF8TIP60 complexes shown in the outlined area in A. The insets on the right show three projections of the single-molecule interaction between PHF8 and TIP60 viewing down the x-, y-, and z-axes, demonstrating that the complexes formed between these two chromatin-modifying enzymes have well-defined spatial relationship. Each dot corresponds to the localization of a single molecule. Scale bars, 50 nm.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Labeling

Journal: eneuro

Article Title: A Neuronal Activity-Dependent Dual Function Chromatin-Modifying Complex Regulates Arc Expression

doi: 10.1523/eneuro.0020-14.2015

Figure Lengend Snippet: Figure 10 PHF8 and TIP60 form a tripartite complex with the splicing factor PSF and associates with newly transcribed nascent RNA. A, A maximum intensity projection of a 3D STORM image of an activated hippocampal neuronal nucleus. Single-molecule imaging of endogenous PHF8 (green), endogenous TIP60 (red), and PTB-associated splicing factor (blue), with the corresponding single- channel views. Each dot corresponds to the localization of a single molecule. Scale bar, 500 nm. B, C, Four representative higher magnification views of the neuronal nucleus depicted in A, showing that PSF (blue) forms a tailing structure within the interface between PHF8 (green) and TIP60 (red) viewed axially B or longitudinally C as a recognizable tripartite complex. Scale bar, 50 nm.

Article Snippet: The primary antibodies were incubated for 1 h at RT in a dilution buffer containing 1:1 block solution and PBS-Triton X solution at the following dilutions: mouse-anti-Arc (C7), 1:300 (Santa Cruz Biotechnology); goat-anti-TIP60 (K17), 1:300; rabbit-anti-TIP60, 1:300 (Novus Biologicals); rabbit-anti-H3K9acS10P, 1:300 (Abcam).

Techniques: Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of H3K9me3 by immunofluorescence staining in WT, LIPT1 −/− H460, and LIPT1 −/− H460 cells stably expressing Myc-LIPT1 cells. ( B ) Representative images and quantification of H3K9me3 by immunofluorescence staining in WT and LIPT1 −/− H157 cells. ( C and D ) Representative images and quantification of in situ proximity ligation assay (PLA, green dots) of interactions between histone H3 and TIP60 interaction (C) and between γH2AX and TIP60 (D) in WT and LIPT1 −/− H460 cells, with or without 10-Gy IR. ( E and F ) Representative images and quantification of in situ PLA (green dots) of interactions between histone H3 and TIP60 interaction (E) and between H2AX and TIP60 (F) in WT and LIPT1 −/− H157 cells with or without 10-Gy IR. For (A) to (F), nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. One-way ANOVA was used for the statistical analyses for (A), unpaired t test was used for (B), and two-way ANOVA was used for (C) to (F). **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: Immunofluorescence, Staining, Stable Transfection, Expressing, In Situ, Proximity Ligation Assay, Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT, LIPT1 −/− H460, and LIPT1 −/− H460 reconstituted Myc-LIPT1 cells. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. ( B ) Quantification of in situ PLA (green dots) of ATM and TIP60 interaction in nonirradiated control and 1 hour after 4 Gy in WT and LIPT1 −/− H157 cells. ( C and D ) Immunoblotting analysis of ATM-pS1981, Ku70, γH2AX, and histone 3 in the soluble nuclear and chromatin fractions of WT and LIPT1 −/− in H460 (C) and in H157 (D) cells, with or without IR (0.5 hours post 10 Gy). Histone H3 and γH2AX served as chromatin markers. LE, long exposure; SE, short exposure. ( E and F ) Immunofluorescence images and quantification of colocalized ATM-pS1981 (red) and γH2AX (green) foci at 0.5 hours post-4 Gy in WT, LIPT1 −/− H460, and Myc-LIPT1–reconstituted H460 cells (E), as well as WT and LIPT1 −/− H157 cells (F). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. ( G and H ) Immunoblot analysis of ATM-pS1981, total ATM, Chk2-pT68, total Chk2, lipoyl-DLAT/DLST, and γH2AX in H460 (G) and H157 (H) cells at 0.5 hour post-10Gy. (A), (B), (E), and (F), quantification was performed on >100 cells per treatment. Two-way ANOVA was used for (A) and (B), one-way ANOVA was used for (E), and unpaired t test was used for (F). **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Control, Staining, Western Blot, Immunofluorescence

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative immunoblots validating siRNA suppression of KDM4B in WT and LIPT1 −/− H460 cells, with GAPDH used as a loading control. ( B ) Representative images and quantification of H3K9me3 by immunofluorescence staining in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( C and D ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 interaction (C), and between ATM and TIP60 (D) in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells, with or without 10- and 4-Gy IR. ( E ) Representative images and quantification of γH2AX foci by immunofluorescence staining in nonirradiated cells and at 6 and 24 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. ( F ) Representative images and quantification of RAD51 foci by immunofluorescence staining in nonirradiated cells and 6 hours after 4-Gy IR in Ctrl and siKDM4B WT and LIPT1 −/− H460 cells. For (B) to (F), nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: Western Blot, Control, Immunofluorescence, Staining, In Situ, Imaging

Journal: Science Advances

Article Title: Lipoylation inhibition enhances radiation control of lung cancer by suppressing homologous recombination DNA damage repair

doi: 10.1126/sciadv.adt1241

Figure Lengend Snippet: ( A ) Representative images and quantification of in situ PLA (green dots) of interactions between γH2AX and TIP60 in WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG and 10-Gy IR. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. Imaging and quantification were performed on >100 cells per treatment. Two-way ANOVA was used for the statistical analyses. **** P < 0.0001. ( B ) Immunoblotting analysis of ATM-pS1981 and histone H3 in the chromatin fractions of WT and LIPT1 −/− in H460 cells with or without 1 mM dimethyl–α-KG, 1 mM α-KG, and 10-Gy IR. Histone H3 was used as marker and internal control for the chromatin fraction. Protein levels of ATM-pS1981 were normalized with internal control histone H3. ( C ) Clonogenic assay of WT and LIPT1 −/− H460 cells with or without 1 mM dimethyl–α-KG after 2, 4, and 6 Gy. The surviving fraction was normalized to the corresponding sham control and survival curves were fitted using the linear-quadratic model. ( D ) Schematic illustrating how LIPT1 deficiency impairs the IR-induced, TIP60-ATM–mediated HR damage repair pathway due to a deficiency in α-KG–dependent demethylation.

Article Snippet: Primary antibodies against histone H3 (819411, RRID:AB_2820127, dilution 1:200, BioLegend, San Diego, CA), TIP60 (NBP2-24613, RRID:AB_3272659, dilution 1:200, Novus Biologicals, Centennial, CO), γH2AX (05-636, RRID:AB_309864, dilution 1:200, MilliporeSigma), and ATM (NB100-309, RRID:AB_2243346, dilution 1:200, Novus Biologicals) in Duolink antibody diluent buffer were then added to the cells and incubated for 3 hours at room temperature.

Techniques: In Situ, Staining, Imaging, Western Blot, Marker, Control, Clonogenic Assay