Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in immortalized MEFs. (A) Cells (WT, MJI-53; KO, MJI-1) were treated for 24 h using 0 or 3 μM aphidicolin. (B) Cells (lane 1–3, WT, MJI-91; lanes 4–6, KO, MJI-186; lanes 7–9, WT, MJI-105; lanes 1–12, KO, MJI-84) were treated for 24 h using 0, 1 or 3 μM aphidicolin. Actin and 53BP1 serve as loading controls. DYNLL1 is used as a surrogate marker for loss of ASCIZ. (C) Quantification of western blot band intensities. An arbitrary unit of 100 represents the average band intensity for the respective phospho-protein in the 3 μM aphidicolin-treated wildtype samples on each membrane. Graphs indicate the mean ± standard error, n = 3. Additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1A and B.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Marker, Western Blot, Membrane

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in primary MEFs. Freshly prepared primary MEFs from a wildtype and an Asciz −/− embryo were treated for 24 h with 0, 1 or 3 μM aphidicolin. For comparison, cultures from the same embryos were also irradiated with 2-Gy and allowed to recover for 30 min. Similar results for another independent pair of wildtype and Asciz −/− primary MEFs are shown in Supplementary Fig. S1C, and additional loading controls for total KAP1, p53 and H2AX are shown in Supplementary Fig. S1D.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Comparison, Irradiation

Journal: DNA Repair

Article Title: ASCIZ/ATMIN is dispensable for ATM signaling in response to replication stress

doi: 10.1016/j.dnarep.2017.06.022

Figure Lengend Snippet: Aphidicolin-induced ATM signaling in human ASCIZ/ATMIN knockout cells. (A, B) BL30 cells were cultured as indicated in the presence or absence of doxycycline to induce a non-targeting control guide RNA or the guide RNA against ASCIZ , and aphidicolin to elicit prolonged DNA replication stress as indicated. For comparison, control guide RNA transduced cells were also irradiated for canonical ATM activation without doxycycline or aphidicolin treatment in panel A. Panel B shows a blot of the same samples except the irradiated control. Additional loading controls for total ATM, KAP1 and p53 are shown in Supplementary Fig. S1E.

Article Snippet: The following antibodies were used for western blots: Actin (EMD Millipore/Merck, MAB1501), ATM (Abcam, 5C2, ab2618), DYNLL1 (Abcam, ab51603), FANCD2 (Abcam, ab178705), 53BP1 (Novus, NB100-304, lot A3), γH2AX (EMD Millipore/Merck, 05-636), H2AX (Abcam, ab20669), KAP1 (Proteintech, 15202-1-AP), M601 (Abcam, ab110411), p53 (Cell Signaling Technology, 1C12; for mouse samples), p53 (Santa Cruz Biotechnology, DO-1, sc-126; for human samples), phospho-CRMP2 (Ser522; ECM Biosciences, CP2191), phospho-ATM (Ser1981; Cell Signaling Technology, 10H11.E12), phospho-KAP1 (Ser824; Bethyl, A300-767A); phospho-p53 (Ser-15; Cell Signaling Technology, 9284); α-tubulin (Sigma-Aldrich, T9026).

Techniques: Knock-Out, Cell Culture, Control, Comparison, Irradiation, Activation Assay

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 3. Identification of PITAR targets. (A) Genomic track for PITAR derived from ChIRP-RNA sequencing using Odd, Even, and LacZ antisense probe. (B) PITAR Pulldown by ChIRP assay was quantified using qRT-PCR. (C) The Venn diagram represents the association of four datasets (ChIRP enriched genes, PITAR positive correlated genes from TCGA, GBM vs. normal upregulated genes from TCGA, and GSC vs. DGC upregulated genes from GSE54791). (D) The selected 15 genes from the Venn diagram are plotted in the scatter plot, and an arrow marked TRIM28 as a selected target. (E) The volcano plot depicts up-regulated (n=420) and down-regulated (n=526) genes upon PITAR knockdown compared to siNT. The gene expression matrix between siPITAR and siNT was used to construct a volcano plot to visualize differentially expressed genes. (F) Gene set enrichment analysis (GSEA) of differentially regulated genes was performed based on PITAR expression level at log2fold >0.58 and p<0.05. (G) The GSEA plots depict the enrichment of p53 up and down target gene sets, results derived from PITAR-silenced U87 cells. Data are shown as mean ± SD (n=3). ***p-value <0.001, **p-value <0.01, *p-value <0.05.

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Derivative Assay, RNA Sequencing, Quantitative RT-PCR, Knockdown, Gene Expression, Construct, Expressing

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 4. PITAR regulates the expression of TRIM28 by physical interaction with TRIM28. (A) The qRT-PCR of PITAR and TRIM28 RNA was performed in ChIRP-RNA pull-down samples. The Probes for Odd and LacZ were used to pull down endogenous PITAR and interacting TRIM28 mRNA in U87 cells. (B) Schematic represents the predicted RNA–RNA interaction between PITAR and the 3′ UTR of TRIM28. (C) RNAScope images of co-localized signals of PITAR (red) and TRIM28 (green) in U87 cells. The panel shows the 3D reconstructed cell images of the merged 2D image (Imaris image analysis software). Yellow dots shown by white arrows depict co-localized PITAR (red) and TRIM28 (green). Magnified co-localized puncta were shown in the inset at the upper right corners, indicated by a white dotted box. The panel shows the RNAScope images of the localization of PITAR (red), TRIM28 (green), and PDE2A (green/red). PDE2A RNA was used as a negative control. The indicative scale bar on the images is 50 µm. (D) Schematic of vector plasmid construct for PITAR OE, ΔPITAR OE, TRIM28 3′ UTR, and ΔTRIM28 3′ UTR. (E) PITAR interaction with the TRIM28 3′ UTR was measured using an in vitro RNA–RNA interaction assay and compared to a panel of control RNAs (PITAR antisense, Fluc control, Bead control). The binding affinity was quantified by qRT-PCR analysis of the TRIM28. Data were normalized to the PITAR-AS control. (F) The Luc activity of TRIM28 3′ UTR was measured after the ectopic expression of PITAR and ΔPITAR in U87 cells using a luciferase reporter assay. (G) Luciferase assay was performed in PITAR silenced U87 cells co-transfected with VC and PITAR OE vector. (H) The Firefly luciferase activity was measured in U87 cells containing a deleted PITAR binding site of TRIM28 3’UTR (ΔTRIM28 3′ UTR), co-transfected with VC and PITAR OE. (I) Relative expression of TRIM28 in PITAR-silenced U87 cells was measured by qRT-PCR. (J) The TRIM28 protein expression was measured by immunoblotting. Data are shown as mean ± SD (n=3) and an unpaired t-test was performed using GraphPad Prism v6 (*p-value <0.05, **p-value <0.01, ****p-value <0.0001). (K) TRIM28 transcript was measured at indicated time points post Actinomycin D (5 μg/ml) treatment in siNT and siPITAR-transfected U87 cells by qRT-PCR (n=3). The log2 ratio of the remaining TRIM28 was plotted using linear regression after normalizing to the 0th hour of the respective condition using GraphPad Prism v6. Data are shown as mean ± SD (**p-value <0.01).

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Expressing, Quantitative RT-PCR, RNAscope, Software, Negative Control, Plasmid Preparation, Construct, In Vitro, Control, Binding Assay, Activity Assay, Luciferase, Reporter Assay, Transfection, Western Blot

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 5. PITAR regulates wild-type p53 protein levels via TRIM28-mediated ubiquitination. (A) Relative expression of PITAR, TRIM28, TP53, and CDKN1A was quantified by qRT-PCR in PITAR-silenced U87 cells compared to siNT. (B) The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in PITAR-silenced U87 cells compared to siNT. (C, D) Cells were transfected with pcDNA3.1-PITAR (PITAR OE)/ empty vector control plasmid (pcDNA3.1) and harvested 48 hr post-transfection for qRT-PCR (PITAR, TRIM28, TP53, and CDKN1A) and immunoblotting with indicated antibodies (TRIM28, p53, and p21). GAPDH served as the control. (E) The Half-life of the p53 protein was measured in PITAR-silencing (siPITAR) and control (siNT) U87 cells with the treatment of cycloheximide (CHX; 50 μg/mL). The relative expression of the remaining p53 was plotted using linear regression after normalizing to the 0th hour of the respective condition. (F) The endogenous level of p53 ubiquitination was measured in pcDNA3.1- PITAR (PITAR OE)/empty vector plasmid (pcDNA3.1) stable U87 cells by p53 immunoprecipitation followed by immunoblotting with the indicated antibodies in the presence and absence of MG132. (G–I) The relative expression of PITAR, TRIM28, and CDKN1A was measured by qRT-PCR in U87/ siPITAR#1 and U87/siNT cells with exogenously overexpressed TRIM28 conditions. (J) The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in U87/siPITAR#1 and U87/siNT cells with exogenously overexpressed TRIM28 condition. (K–M) The relative expression of PITAR, TRIM28, and CDKN1A was measured by qRT-PCR in U87/shTRIM28 and U87/shNT cells with exogenously overexpressed PITAR conditions. (N) The protein expression of TRIM28, p53, and p21 was measured by immunoblotting in U87/shTRIM28 and U87/shNT cells with exogenously overexpressed PITAR conditions. Data are shown as mean ± SD (n=3) and an unpaired t-test was performed using GraphPad Prism v6 (*p-value <0.05, **p-value <0.01,***p-value <0.001, ****p-value <0.0001).

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Control, Immunoprecipitation

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 6. DNA damage-induced PITAR diminishes the DNA damage response by p53 through TRIM28. (A) The relative expression of CDKN1A was measured in the presence and absence of Adriamycin by qRT-PCR in PITAR OE/VC U87 cells. (B) The protein expression of TRIM28, p53, ac-p53, and p21 was measured in the presence and absence of Adriamycin by immunoblotting in PITAR OE/VC U87 cells. GAPDH served as the control. (C) The viable cell count was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. (D) The cell cycle analysis was performed in the presence and absence of Adriamycin in PITAR OE/VC U87 cells. (E) The relative expression of PITAR, TRIM28, TP53, and CDKN1A was measured by qRT- PCR in Ad-p53 and Ad-GFP-infected U87 cells. (F) The protein expression of TRIM28 and p53 was measured by immunoblotting in Ad-p53 and Ad-GFP- infected U87 cells. (G) The relative expression of PITAR, TRIM28, and CDKN1A was measured in the presence and absence of Adriamycin by qRT-PCR in U87 cells. (H) The immunoblot depicting the expression of TRIM28, p53, and p21 upon treatment of Adriamycin. (I, J) The qRT-PCR was performed to measure the relative expression of PITAR and TRIM28 in the p53 knockdown condition upon Adriamycin treatment. (K) The relative expression of PITAR and TRIM28 was measured by qRT-PCR in Adriamycin-treated PITAR-silenced U87 cells. Data are shown as mean ± SD (n=3) and an unpaired t-test was performed using GraphPad Prism v6 (*p-value <0.05, **p-value <0.01,***p-value <0.001, ****p-value <0.0001).

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Cell Counting, Cell Cycle Assay, Infection, Knockdown

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 7. PITAR promotes glioma tumor growth and resistance to Temozolomide chemotherapy. (A) Mice (NIH nu/nu) were injected with siNT/ siPITAR#1 transfected U87-Luc cells (0.3x106 cells/mice), and tumors were allowed to grow for 50 days (n=10), and the luminescence imaging was performed using the IVIS instrument (Perkin Elmer IVIS system). (B) H&E staining was performed in formalin-fixed tumor-bearing (siNT and siPITAR#1) mouse brain sections. (C) The tumor growth curve of siNT and siPITAR#1 tumor-bearing mice was quantified over time using IVIS. The difference between groups was statistically analyzed by ANOVA with Tukey’s multiple comparisons test (*p<0.05).(D) The Kaplan–Meier graph shows the survival of mice-bearing tumors formed by siNT and siPITAR#1 cells (N=10/group), statistical differences were calculated with the Log-rank (Mantel-Cox) test (*p<0.05). (E) The mmunohistochemistry assay was performed to show the TRIM28 protein expression in the tumor tissue section derived from U87-Luc/ siNT and U87-Luc/siPITAR#1 tumors. The Green color represents the TRIM28 protein, and the blue depicts the nucleus stained with DAPI. Scale bar = 100 μm. (F) Mice (NIH nu/nu) were injected with U87-Luc/PITAR OE and U87-Luc/VC cells (0.3x106 cells/mice, n=10), and tumors were allowed to grow for 30 days. The tumor-bearing mice were treated with 100 mg/kg TMZ in 25% DMSO saline solution after 11 days by intraperitoneal injection for one week, and the luminescence imaging was performed using the IVIS instrument. (G) The tumor growth curve of VC, PITAR OE, VC +TMZ, and PITAR OE +TMZ tumor-bearing mice was quantified over time using IVIS. The difference between groups was statistically analyzed by ANOVA with Tukey’s multiple comparisons test (*p<0.05, ***p<0.001). (H) Mice (NIH nu/nu) were injected with U87-Luc/PITAR OE +shNT, U87-Luc/VC +shNT, U87-Luc/ PITAR OE +shTRIM28 and U87-Luc/VC +shTRIM28 cells (0.3x106 cells/mice), and tumors were allowed to grow for 50 days. (I) The tumor growth curve of VC +shNT, PITAR OE +shNT, VC +shTRIM28, and PITAR OE +shTRIM28 tumor-bearing mice (n=10) was quantified over time using IVIS. Luminescence was evaluated twice per 10 days and before sacrifice. Bars indicate standard error and the difference between groups was statistically analyzed by ANOVA with Tukey’s multiple comparisons post-test (*p<0.05, **p<0.01).

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Injection, Transfection, Imaging, Staining, Expressing, Derivative Assay, Saline

Journal: eLife

Article Title: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

doi: 10.7554/elife.88256

Figure Lengend Snippet: Figure 8. Proposed working model of this study. PITAR inhibits p53 by binding and stabilizing TRIM28 mRNA.

Article Snippet: The TRIM28 3' UTR Luc vector plasmid was purchased from Origen, USA (# SC202088).

Techniques: Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 1. RNA viral infection induces KAP1 expression and SUMOylation. (A and B) Western blot analysis of KAP1 expression in mouse PMs (A) or THP-1 cells (B) infected with VSV or SeV for the indicated time periods. (C) Western blot analysis of KAP1 expression in mouse PMs stimulated with IFN- b for the indicated time periods. (D) Western blot analysis of KAP1 expression in Ifnar11/1 or Ifnar1/ PMs infected with SeV for the indicated time peri- ods. (E and F) Western blot analysis of KAP1 expression in mouse PMs pretreated with JSH-23 or itacitinib for 24 h and then infected with SeV. (G) Western blot analysis of KAP1 SUMOylation in HEK293T cells cotransfected with plasmids encoding FLAG-KAP1, HA-SUMO-1, and His-UBC9 and then infected with SeV. All experiments were repeated three times.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Infection, Expressing, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 2. KAP1 inhibits RNA viralinduced IFN-b activation. (A) Luciferase analysis of IFN-b reporter activation in HEK293T cells trans- fected with IFN-b reporter plasmid with empty control, TRIM28 (KAP1), TRIM24, TRIM33, or TRIM66 expression plasmid and then infected with SeV for 12 h. (B) Luciferase analysis of IFN-b reporter activation in HEK293T cells transfected with IFN-b reporter plasmid, together with empty control plasmid or KAP1 expression plasmids, and then infected with VSV for 12 h. (C) Western blot analysis of KAP1 expression in mouse PMs from WT or CKO mice. Data shown are means ± SD [n 5 6 in (A), and n 5 5 in (B)]. All experiments were repeated three times. **p < 0.01, ***p < 0.001, by Student t test.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Activation Assay, Luciferase, Plasmid Preparation, Control, Expressing, Infection, Transfection, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 3. KAP1 inhibits RLR pathway. (A and B) ELISA analysis of IFN-b and TNF-a secretion in PMs from WT or CKO mice infected with SeV (A) or VSV (B) for the indicated time periods. (C and D) PMs from WT or CKO mice were infected with SeV (C) or VSV (D) for the indicated time periods. RT-PCR analysis of Ifnb mRNA expression. (E and F) PMs from WT or CKO mice were infected with SeV (E) or VSV (F) for 12 h. RT-PCR analysis of Ifna4 mRNA expression. (GJ) Western blot analysis of IRF3 phosphorylation in WT or KAP1 CKO mouse PMs infected with SeV (G) or VSV (H) trans- fected with poly(I:C) HMW (I) or poly(I:C) LMW (J) for the indicated time periods. (K) RT-PCR analysis of IFNB mRNA expression in control (CTRL) siRNA or KAP1 siRNAtransfected THP-1 cells, followed by SeV infection for 12 h. (L and M) Western blot analysis of IRF3 phosphorylation in Ctrl siRNA or KAP1 siRNA-1transfected THP-1 cells, followed by SeV (L) or VSV (M) infection for the indicated time periods. (N) RT-PCR analysis of Ifnb mRNA expression in Ctrl siRNA or Kap1 siRNA-1transfected Ifnar1/ PMs, followed by SeV or VSV infection for 12 h. (O and P) Western blot analysis of IRF3 phosphorylation in Ctrl siRNA or Kap1 siRNA-1transfected Ifnar1/ PMs, followed by SeV (O) or VSV (P) infection. Data shown are means ± SD [n 5 3 in (AF, K, and N)]. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, by Student t test.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 4. Kap1 deficiency enhances anti-RNA viral innate responses. (A and B) Western blot analysis of STAT1 phosphorylation in PMs from WT or CKO mice infected with SeV (A) or VSV (B) for the indicated time periods. (C and D) RT-PCR analysis of Ifit1, Ifit2, and Mx1 expression in mouse PMs from WT or CKO mice infected with SeV (C) or VSV (D) for 12 h. (E) RT-PCR analysis of the replication of VSV RNA in PMs from WT or CKO mice infected with VSV for 12 h. (F) The 50% tissue culture infectious dose assay analysis VSV loads in mouse PMs. (G and H) WT or CKO mice were i.p. injected with VSV for 8 h. ELISA analysis of IFN-b secretion in serum (G). RT-PCR analysis of the replication of VSV RNA (H). Data shown are means ± SD [n 5 3 in (CF), n 5 6 in (G), and n 5 5 in (H)]. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, by Student t test.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Western Blot, Phospho-proteomics, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing, Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 5. KAP1 promotes repressive histone marks establishment at Ddx58 and Ifih1 promoters. (AC) Mouse PMs were injected with VSV for 8 h and then prepared for ChIP-seq or ChIP-qPCR assay using anti-KAP1 Ab. (A) Distribution of TRIM28-binding peaks in mouse PMs. (B) Type I IFN signal- ingrelated genes enriched with KAP1 binding peaks at their promoter regions analyzed from ChIP-seq. (C) University of California Santa Cruz browser view of KAP1 binding peaks at the Ddx58 or Ifih1 gene locus. (D) ChIP-qPCR analysis of KAP1 binding at Ddx58 or Ifih1 gene loci in VSV-uninfected and VSV-infected mouse PMs. Irf4 served as a negative control. (EG) WT or CKO mouse PMs were injected with VSV for 8 h and then prepared for ChIP- qPCR assay performed by anti-H3K9me3 Ab (E), H3K4me3 Ab (F), or pol II Ab (G). Irf4 served as a negative control. Data shown are means ± SD [n 5 3 in (DG)]. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, by Student t test. ns, not significant.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Injection, ChIP-sequencing, ChIP-qPCR, Binding Assay, Infection, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: KAP1-Mediated Epigenetic Suppression in Anti-RNA Viral Responses by Direct Targeting RIG-I and MDA5.

doi: 10.4049/jimmunol.2100342

Figure Lengend Snippet: FIGURE 6. KAP1 enhances RIG-I and MDA5 expression. (A) Primers used to detect primary transcripts of Ddx58 and Ifih1. (B and C) RT-PCR analysis of primary transcripts of Ddx58 (B) and Ifih1 (C) in WT or CKO mouse PMs infected with SeV or VSV for 8 h. (D and E) RT-PCR analysis of Ddx58 (D) and Ifih1 (E) mRNA expression in WT or CKO mouse PMs infected with SeV or VSV for 12 h. (F and G) Western blot analysis of RIG-I and MDA5 expres- sion in WT or CKO mouse PMs infected with SeV (F) or VSV (G). (H) RT-PCR analysis of Ddx58 and Ifih1 mRNA expression in control siRNA or Kap1 siRNA-1transfected Ifnar1/ PMs, followed by SeV infection for 12 h. (I) Western blot analysis of RIG-I and MDA5 expression in HEK293T cells trans- fected with empty control, KAP1, KAP1 DRING, or KAP1 C651 plasmid. Data shown are means ± SD [n 5 3 in (BE and H)]. All experiments were repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, by Student t test.

Article Snippet: KAP1 plasmid was purchased from OriGene (SC321588) and subcloned into FLAG tag to generate FLAG-KAP1.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Control, Plasmid Preparation

Journal: Molecular Cancer Research

Article Title: Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

doi: 10.1158/1541-7786.mcr-10-0042

Figure Lengend Snippet: FIGURE 2. Reciprocal coimmunoprecipitation of ErbB4 and Kap1 in breast cancer cell lines. A, coimmunoprecipitation from NRG-treated BT-474 cells. Five hundred micrograms of BT-474 cell lysate were immunoprecipitated with 2 μg of total rabbit IgG, anti-Kap1, or anti-ErbB4. Immune complexes were isolated using protein A/G beads and analyzed on 4% to 12% NuPAGE Bis-Tris gels. Portions of the lysates were analyzed directly in lanes 5 to 8. Immunoblots were probed with the reciprocal antibodies to those used for immunoprecipitation. Asterisks mark the full-length and 80-kDa cleaved forms of ErbB4 (lanes 2, 7, and 8) or Kap1 (lanes 4-6). NS, nonspecific band on ErbB4 blots. B, coimmunoprecipitation of ErbB4 and Kap1 from NRG-treated T-47D and MDA-MB453 cells. Portions of the lysates were analyzed directly without immunoprecipitation in lanes 9 to 12. C, HEK293T cells were transfected with 1 μg each of the plasmids encoding full-length ErbB4 and Flag-Kap1. After 24 h, the cells were switched to starvation medium (0.1% FBS in DMEM) for 16 h and then mock stimulated with DMSO vehicle (TPA−) or with 100 ng/mL TPA (TPA+) in DMSO. Lysates were immunoprecipitated with anti-Kap1, anti-ErbB4, or control IgG (Ig). Kap1 and ErbB4 were identified by immunoblotting, with ∼100-kDa Kap1 and full-length and cleaved ErbB4 forms. Blotting for GAPDH (not shown) verified even loads for 10% input tracks. Data represent at least two biological replicates in identical format plus two similar biological replicates (A and B).

Article Snippet: The product was digested and ligated into a leaved pBABE retroviral vector to generate s80 E. The plasmids containing the GAL4 DNA binding in fused with ERBB4 cytoplasmic tail fragment ) and CTF ΔK with deleted kinase domain were y provided by Marius Sudol (Geisinger Health Weis Center for Research, Danville, PA; ref. 13). uman Krab-associated protein 1 (Kap1) plasmid urchased from Origene.

Techniques: Immunoprecipitation, Isolation, Western Blot, Transfection, Control

Journal: Molecular Cancer Research

Article Title: Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

doi: 10.1158/1541-7786.mcr-10-0042

Figure Lengend Snippet: FIGURE 3. Effect of ErbB4 kinase activity and ICD isoform on interaction with Kap1. Truncated V5-tagged ErbB4 isoforms CYT-1, kinase-deficient CYT-1 (CYT-1 KD), CYT-2, or CYT-2 KD were transiently coexpressed with Flag-tagged Kap1 in COS-7 cells that were then stimulated with 100 ng/mL TPA. Protein extracts were immunoprecipitated using antibodies against the epitope tags and immunoblotted as marked. Portions of the lysates were analyzed directly by immunoblotting for V5, Flag, and GAPDH without immunoprecipitation. Data represent two biological replicates in identical format plus two similar biological replicates.

Article Snippet: The product was digested and ligated into a leaved pBABE retroviral vector to generate s80 E. The plasmids containing the GAL4 DNA binding in fused with ERBB4 cytoplasmic tail fragment ) and CTF ΔK with deleted kinase domain were y provided by Marius Sudol (Geisinger Health Weis Center for Research, Danville, PA; ref. 13). uman Krab-associated protein 1 (Kap1) plasmid urchased from Origene.

Techniques: Activity Assay, Immunoprecipitation, Western Blot

Journal: Molecular Cancer Research

Article Title: Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

doi: 10.1158/1541-7786.mcr-10-0042

Figure Lengend Snippet: FIGURE 4. ErbB4 and Kap1 interact with MDM2. COS-7 cells were transiently transfected with vector alone (M) or vectors encoding truncated CYT-2 or CYT-2 KD, Flag-tagged Kap1, and T7-tagged MDM2. Nuclear and cytoplasmic fractions were immunoprecipitated with anti-Kap1 (A) or anti-ErbB4 (B) and immunoblotted with anti-ErbB4, Kap1, or MDM2 antibodies. The blot in A was stripped and reprobed with anti-Kap antibody. The blot in B was stripped and reprobed with anti-ErbB4 antibodies. A separate blot of the same immunoprecipitations was probed with anti-ErbB4 antibody (A) to confirm the Kap1/ErbB4 interaction. Similarly, the blot in B was probed with anti-Kap antibody to confirm the ErbB4/Kap1 interaction. C, portions of the nuclear and cytoplasmic extracts were analyzed without immunoprecipitation, as marked. Data represent two biological replicates in identical format plus two similar biological replicates.

Article Snippet: The product was digested and ligated into a leaved pBABE retroviral vector to generate s80 E. The plasmids containing the GAL4 DNA binding in fused with ERBB4 cytoplasmic tail fragment ) and CTF ΔK with deleted kinase domain were y provided by Marius Sudol (Geisinger Health Weis Center for Research, Danville, PA; ref. 13). uman Krab-associated protein 1 (Kap1) plasmid urchased from Origene.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Molecular Cancer Research

Article Title: Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

doi: 10.1158/1541-7786.mcr-10-0042

Figure Lengend Snippet: FIGURE 5. Effect of MDM2 expression on ErbB4/Kap1/p53 interactions. A and B, ErbB4 and Kap1. HEK293T cells were infected with pSiren or pSiren shMDM2 virus. After 24 h, the cells were transfected with pcDNA3.1 V5/His or s80 Cyt2 in the same vector. At 72 h after infection, the cells were harvested and total protein was extracted. A, immunoblots for endogenous MDM2, Kap1, and GAPDH and ectopic s80 ErbB4 in cell lysates. B, anti-V5 immunoprecipitation, with ErbB4 or Kap1 immunoblot from the samples analyzed in A. C, ErbB4, Kap1, and p53. COS-7 cells were transfected with vector (V; lanes 1 and 5); with 2 μg of truncated CYT-1 ErbB4 and 2 μg of Flag-tagged Kap1 (EK; lanes 2 and 6); with 2 μg of truncated CYT-1 ErbB4, 2 μg of Flag-tagged Kap1, and 2 μg of MDM2 (EKM; lanes 3 and 7); and with 2 μg of truncated CYT-1 ErbB4, 2 μg of Flag-tagged Kap1, and 4 μg of MDM2 (EKM; lanes 4 and 8). Cells were treated with 10 μmol/L MG132 for 3.5 h and stimulated with TPA for 30 min before harvest at 48 h. Data represent two biological replicates in identical format plus two similar biological replicates (A and B).

Article Snippet: The product was digested and ligated into a leaved pBABE retroviral vector to generate s80 E. The plasmids containing the GAL4 DNA binding in fused with ERBB4 cytoplasmic tail fragment ) and CTF ΔK with deleted kinase domain were y provided by Marius Sudol (Geisinger Health Weis Center for Research, Danville, PA; ref. 13). uman Krab-associated protein 1 (Kap1) plasmid urchased from Origene.

Techniques: Expressing, Infection, Virus, Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: Molecular Cancer Research

Article Title: Interactions of ErbB4 and Kap1 Connect the Growth Factor and DNA Damage Response Pathways

doi: 10.1158/1541-7786.mcr-10-0042

Figure Lengend Snippet: FIGURE 8. Kap1 knockdown increases ErbB4 RNA and protein expression. MCF-7 cells were infected with shKap1 virus or control virus for 3 d. Total RNA and protein were extracted using the Qiagen RNeasy Mini Kit. A, RNA was analyzed for the presence of Kap1 and ErbB4 RNA using the TaqMan assay system and normalized to GAPDH expression, which was constant. Bars, SD. A, each analysis was done in triplicate with three biological replicates. B, expression of Kap1 and ErbB4 with Kap1 knockdown (left) or control (right). Data in B represent three biological replicates in identical format.

Article Snippet: The product was digested and ligated into a leaved pBABE retroviral vector to generate s80 E. The plasmids containing the GAL4 DNA binding in fused with ERBB4 cytoplasmic tail fragment ) and CTF ΔK with deleted kinase domain were y provided by Marius Sudol (Geisinger Health Weis Center for Research, Danville, PA; ref. 13). uman Krab-associated protein 1 (Kap1) plasmid urchased from Origene.

Techniques: Knockdown, Expressing, Infection, Virus, Control, TaqMan Assay