Journal: Nature

Article Title: DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis

doi: 10.1038/s41586-020-2041-2

Figure Lengend Snippet: a, b, Immunofluorescence staining of endogenous KU86 (a) and DNA-PKcs (b) in U2OS cells. DDX21 RNA helicase is used as a positive control for nucleoli. The CSK buffer contains Triton X-100 for pre-extraction before fixation (see Methods). When indicated, the cells were treated with 50 nM ActD for 1 h before pre-extraction, fixation and staining. c, Localization of ectopically expressed GFP-tagged KU70 in mouse ES cells. a–c, n = 3 biologically independent experiments. d, U3 ChIRP-qRT–PCR analysis from HeLa cells. Enrichment levels, relative to input samples, of the U3, 7SK, 18S, and RMRP RNAs were assessed from experimental (−RNase A) or control (+RNase A) ChIRP samples. Data are from two independent biological replicates. e, DNA-PK was also recovered from U3 ChIRP-MS in IMR90 cells. Peptide spectral match (PSM) counts for control (RNase A) and experimental (U3) samples are shown. n = 2 biological replicates.

Article Snippet: Fixed cells were then incubated with primary antibodies in 3% BSA for 1 h at 25 °C, including mouse anti-human KU86 (ThermoFisher, MA5–12933, 1:100), rabbit anti-human DDX21 (Novus, NB100–1718, 1:500) or anti-DNA-PKcs (ThermoFisher, Ab-4(cocktail)), followed by fluorophore-conjugated secondary antibodies (Alexa Fluor 488-conjugated anti-rabbit, Alexa Fluor 594-conjugated anti-rabbit, and cyanine3-conjugated anti-mouse, Invitrogen, 1:500) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Positive Control, Extraction, Quantitative RT-PCR, Control

Journal: International journal of molecular sciences

Article Title: Epigenetic Silencing of miR-218-5p Modulates BIRC5 and DDX21 Expression to Promote Colorectal Cancer Progression.

doi: 10.3390/ijms26094146

Figure Lengend Snippet: Figure 6. Validation of selected miR-218-5p Gene Targets. (A) Validation of BIRC5 and DDX21 as bona fide gene targets for miR-218-5p in CRC. Two-tailed t-test was used to compare different groups. ** p < 0.005, *** p < 0.0005, **** p < 0.00005. (B) Western blot showing DDX21 protein expression in miR-218-5p overexpressing compared to control HT-29 and HCT116 cells. Quantification of DDX21 protein expression normalized to ACTB is shown in the right panel. (C) Gene effect score based on CRISPR-Cas9 screen data in 40 CRC cell models from the DepMap database.

Article Snippet: After 1 h, primary DDX21 Antibody (D-8) (Santa Cruz Biotechnology, Cat. No. sc-376953, Dallas, TX, USA) was diluted in TBST 1:250 and incubated overnight at 4 ◦C on a rocker and then for 1 h in anti-mouse IgG, HRP-linked Antibody 1:2000 (Cell Signaling Technology, Cat. No. 7076, Danvers, MA, USA) the next day.

Techniques: Biomarker Discovery, Two Tailed Test, Western Blot, Expressing, Control, CRISPR

Journal: International journal of molecular sciences

Article Title: Epigenetic Silencing of miR-218-5p Modulates BIRC5 and DDX21 Expression to Promote Colorectal Cancer Progression.

doi: 10.3390/ijms26094146

Figure Lengend Snippet: Figure 7. Suppression of SLIT2 and SLIT3 miR-218 Host Genes in CRC. (A) Genomic location of miR- 218-1 and miR-218-2 within the SLIT2 and SLIT3 genomic region on chromosome 4 and 5, respectively. (B) Correlation plot between miR-218-5p and SLIT2 (left) and SLIT3 (right) in a large cohort of COAD (n = 450) from the ENCORI project. (C) Downregulation of SLIT2 (left) and SLIT3 (right) in COAD (n = 275) compared to normal colon tissue (n = 349) from GEPIA2 database. T: tumor, N: normal. * p < 0.05. (D) Methylation analysis of SLIT2 and SLIT3 promoters using bisulfite conversion and NGS in a panel of CRC cell models (HCT116, HT-29, SW-480, LoVo, and DLD-1) compared to the MCF10A normal epithelial cells. (E) Schematic representation illustrating ( ) downregulation of SLIT2 and SLIT3 in CRC to lead to miR-218-5p suppression ( ), thus promoting tumorigenesis due to lifted suppression ( ) of BIRC5, DDX21, and other gene targets identified in the current study.

Article Snippet: After 1 h, primary DDX21 Antibody (D-8) (Santa Cruz Biotechnology, Cat. No. sc-376953, Dallas, TX, USA) was diluted in TBST 1:250 and incubated overnight at 4 ◦C on a rocker and then for 1 h in anti-mouse IgG, HRP-linked Antibody 1:2000 (Cell Signaling Technology, Cat. No. 7076, Danvers, MA, USA) the next day.

Techniques: Methylation

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A) Representative immunofluorescence images show a HeLa cell transfected with HA-AnkG (red) and GFP-DDX21 (green). (B) HEK293T cells were transiently co-transfected with GFP-tagged AnkG-variants and HA-DDX21. Proteins were precipitated using GFP-trap. Western blot analysis was used to detect DDX21 (anti-HA) and GFP or GFP-tagged AnkG variants (anti-GFP) in the lysates and in the precipitates (IP). A representative western blot out of three independent experiments with similar results is depicted.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Immunofluorescence, Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A and B) GFP or GFP-AnkG 1-28 were transiently expressed in HeLa cells. The cells were either not treated or treated for 4 hours with 0.1 μM staurosporine and stained with an antibody against endogenous DDX21. The subcellular localization of DDX21 was analyzed by epifluorescence microscopy. (A) A representative immunofluorescence image is shown. Upper rows without treatment, lower rows with staurosporine-treatment. (B) The DDX21 localization was analyzed from 50 GFP-expressing cells treated with staurosporine in each of three independent experiments. Error bars indicate ± SD. * p < 0.05. (C) HEK293T cells were transfected with plasmids encoding GFP or GFP-NLS-AnkG. RNA-immunoprecipitation was performed using an anti-GFP antibody. The 7SK RNA was reverse transcribed and a qRT-PCR was performed using specific primers. Data presented as percentage of input bound by GFP or GFP-NLS-AnkG (n = 3). (D) HEK293T cells were co-transfected with HA-tagged AnkG variants and GFP-LARP7 or GFP as control. Proteins were precipitated using GFP-trap. Western blot analysis was used to detect AnkG variants (anti-HA), GFP-LARP7 or GFP (anti-GFP) and endogenous CDK9 (anti-CDK9) in the lysates and in the precipitates (IP). Representative immunoblots out of three independent experiments with similar results are depicted.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Staining, Epifluorescence Microscopy, Immunofluorescence, Expressing, Transfection, RNA Immunoprecipitation, Reverse Transcription, Quantitative RT-PCR, Control, Western Blot

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: RNAs identified in the RIP-Seq analysis. The average fragments per kilobase million (FPKM) from three independent experiments are shown. The 15 genes with most enriched RNAs and five additional RNAs known or predicted to interact with DDX21 are depicted. RNAs with confirmed interaction with AnkG are high-lighted in bold letters and interaction with DDX21 (deduced from RNAct database) is indicated in italic letters.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques:

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A) GFP and GFP-tagged AnkG 1-28 -mutations were transiently co-expressed with HA-DDX21 in HEK293T cells. Proteins were precipitated using GFP-trap. Western blot analysis was used to detect AnkG 1-28 mutations (anti-GFP) and HA-DDX21 (anti-HA) in the lysates (Pre-IP) and in the precipitates (IP). One out of three independent experiments with similar results is shown. (B) GFP and GFP-tagged AnkG 1-28 -mutations were transiently co-expressed with HA-LARP7 in HEK293T cells. Proteins were precipitated using GFP-trap. Western blot analysis was used to detect AnkG 1-28 mutations (anti-GFP) and HA-LARP7 (anti-HA) in the lysates and in the precipitates (IP). A representative blot is depicted from three independent experiments with similar results. (C) HEK293T cells were transfected with GFP or GFP-tagged AnkG-variants. RNA-immunoprecipitation was performed using an anti-GFP antibody. The 7SK RNA was reverse transcribed and a qRT-PCR was performed using specific primers. Data presented as percentage of input bound by GFP or GFP-tagged AnkG variants (n = 5). Error bars indicate ± SD. * p < 0.05, n. s. = not significant. (D) HeLa cells were transiently transfected with plasmids encoding GFP or GFP-tagged AnkG 1-28 -mutations. Cells were treated with 0.1 μM staurosporine for 4 h, fixed and the DNA was stained with DAPI. The morphology of the nuclei of 100 GFP-expressing cells were scored in three independent experiments. Error bars indicate ± SD. * p <0.05, ** p < 0.01, n. s. = not significant.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Western Blot, Transfection, RNA Immunoprecipitation, Reverse Transcription, Quantitative RT-PCR, Staining, Expressing

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: Two HEK293T mutant cell lines lacking the 7SK RNA (ΔLARP7 1–11 and ΔLARP7 3–6) and a respective control were co-transfected with HA-tagged DDX21 and GFP, GFP-NLS-AnkG or GFP-AnkG 1-28 . Proteins were precipitated using GFP-trap. Western blot analysis was used to detect GFP or GFP-tagged AnkG variants (anti-GFP) and HA-DDX21 (anti-HA). Representative immunoblots out of three independent experiments with similar results are shown.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Mutagenesis, Control, Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A) HeLa cells were either not treated, treated with non-target siRNA or with a range of DDX21 siRNA concentrations for 48 h. Cell lysates were analyzed by immunoblot analysis with an anti-DDX21 and an anti-GAPDH antibody as loading control. A representative immunoblot out of two independent experiments with similar results is shown. (B) HeLa cells either not treated, treated with non-target siRNA or with 2 nM DDX21 siRNA for 24 h, were transfected with plasmids encoding GFP, GFP-AnkG 1-28 and GFP-AnkG 1-28 R27S . At 20 h post-transfection, cells were treated with 0.1 μM staurosporine for 4 h, fixed and the DNA was stained with DAPI. The morphology of the nuclei of 100 GFP-expressing cells were scored in four independent experiments. Error bars indicate ± SD. ** p < 0.01, *** p < 0.001, n. s. = not significant. (C—E) HEK293T cells either treated with non-target siRNA or with 10 nM DDX21 siRNA for 24 h were transfected with plasmids encoding GFP, GFP-AnkG 1-28 and GFP-NLS-AnkG. (C) At 24 h post-transfection DDX21 knock-down was analyzed by immunoblot analysis using an anti-DDX21 antibody and an anti-GAPDH antibody as loading control. (D) RNA-immunoprecipitation was performed using an anti-GFP antibody. The 7SK RNA was reverse transcribed and a qRT-PCR was performed using specific primers. Data presented as percentage of input bound by GFP or GFP-tagged AnkG variants from one representative experiment out of three independent experiments. (E) At 24 h post-transfection proteins were precipitated using GFP-trap. Western blot analysis was used to detect GFP and GFP-AnkG 1-28 (anti-GFP), endogenous LARP7 (anti-LAPR7) and endogenous DDX21 to confirm knock-down. Representative immunoblots out of three independent experiments with similar results are shown.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Western Blot, Control, Transfection, Staining, Expressing, Knockdown, RNA Immunoprecipitation, Reverse Transcription, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A-C) HeLa cells were either uninfected (mock), infected with wild-type C . burnetii (WT), C . burnetii Δ ankG (Δ ankG ) or C . burnetii Δ ankG ::AnkG (compl). 24 hours post-infection cells were either treated with non-target siRNA or with DDX21 siRNA for 48 hours. (A) Cell lysates were subjected to western blot analysis using antibodies directed against DDX21, bacterial HSP60 for bacterial loading control and actin as loading control. (B) Cells were treated with 0.1 μM staurosporine for 4 h, fixed and stained with an antibody against LAMP-2. The host cell nucleus and bacterial DNA was stained with DAPI. The morphology of the nuclei of 100 infected cells was scored in three independent experiments. Error bars indicate ± SD. ** p < 0.01, *** p < 0.001 n. s. = not significant. (C) Representative immunofluorescence images are shown. Scale bar 10 μm.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Infection, Western Blot, Control, Staining, Immunofluorescence

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: (A) Schema of AnkG activity. In addition to the properties mentioned in , AnkG interact with the host cell protein DDX21 and with the host cell RNA 7SK. The binding is prevented by mutation of amino acid 27. (B) In the absence of AnkG DDX21 migrates into the nucleoplasm after apoptosis induction. In the presence of AnkG an apoptotic signal results in migration of AnkG into the host cell nucleoli where it binds DDX21. AnkG-DDX21 interacts with the 7SK RNA. This induces the release of P-TEFb (comprising CDK9 and Cyclin T1) and HEXIM from the 7SK snRNP. Released CDK9 phosphorylates NELF, DSIF and the C-terminal domain of Pol II. These promoter-proximal phosphorylation events allow Pol II pause release and transcription of target genes. The presence of AnkG results in transcriptional modulation specifically of genes involved in regulation of apoptosis, trafficking or transcription. As consequence, cells containing AnkG will survive a pro-apoptotic signal.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: Activity Assay, Binding Assay, Mutagenesis, Migration, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

doi: 10.1371/journal.ppat.1010266

Figure Lengend Snippet: Used plasmids.

Article Snippet: Proteins were detected using specific primary antibodies against CDK9 (Cell Signaling), DDX21 (Abcam), GFP (Thermo Fisher Scientific), LARP7 (Abcam), bacterial HSP60 (Enzo), actin (Sigma), GAPDH (Cell Signaling), HA (Roche) and HRP-conjugated secondary antibodies (Dianova).

Techniques: