Journal: BMC cancer

Article Title: Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line.

doi: 10.1186/1471-2407-14-434

Figure Lengend Snippet: Figure 2 Assessment of cellular radiosensitivity in the colony formation assay. A. Cellular radiosensitivity of p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by the colony formation assay after irradiation at doses of 0, 0.1, 0.25, 0.5 or 1Gy. The surviving fraction is presented as the mean value with SEM from at least 3 independent experiments. B. Induction of PARP1 cleavage after irradiation with 2 Gy in p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by immunoblotting of cleaved PARP1 (89 kDa) and total PARP1 (116 kDa). C. Immunoblot analysis of radiation-induced ATM signalling in HCC1395 cells compared with MCF10A. Cells were untreated or irradiated with 0.5, 1, 2, 4 or 6 Gy as indicated. Protein extracts were prepared 30 min after irradiation and were analysed through Western blotting for their immunoreactivity towards the phosphorylated forms of SMC1 (pSer966, top panel), KAP1 (p824, middle panel), and CHEK2 (pSer19, bottom panel). β-actin served as the loading control in each experiment.

Article Snippet: Antibodies to NBN, SMC1pS966, KAP1-pS824 were obtained from Novus Biologicals (rabbit polyclonal); anti CHEK2-pS19 from Cell Signaling (rabbit polyclonal); anti RAD50 (mouse monoclonal) from Abcam; anti MRE11 (mouse monoclonal 12D7) from GeneTex; PARP1 and cleaved PARP1 (rabbit polyclonal) from Cell Signaling, and anti β-Actin (mouse monoclonal) from Sigma.

Techniques: Colony Assay, Mutagenesis, Irradiation, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Functional siRNA screen reveals candidate mitotic phosphatases. (A) Screen schematic. 801 siRNAs targeting 267 phosphatases were used in the screen. (B) Nuclear morphology of cells transfected with indicated siRNAs and treated with 100 nm taxol for 24 h 2 d after siRNA transfection. Negative control cells arrest in mitosis when exposed to taxol. Multinucleation resulting from SAC failure occurs in cells transfected with siRNAs targeting CDKN3, ANP32A, INPP5E, 5NT, SAC1, and PP1M as well as MAD2 (positive control). CDC25A knockdown results in premitotic arrest. (C) Quantification of screen results. Broken lines: 95% confidence interval. P-values were calculated using one-way ANOVA. n = 3 counts for each siRNA (a single representative experiment out of two repeats). Error bars represent mean values ± SEM. (D) Subquantification of phenotypes into premitotic arrest versus multinucleation. Knockout of all screen hits except for CDC25A results in SAC failure. Error bars represent mean values of three independent counts ( n = 3). (E) Generation of cell lines expressing tetracycline-inducible shRNAs and GFP. Western blots show target knockdown as a function of time in response to shRNA induction. The SAC failure in MAD2 and CDKN3 shRNA cells is indicated by a decreased phospho-H3 fraction in cells exposed to 100 nm taxol for 24 h after 72 h of tetracycline induction (P < 0.0001 for MAD2 and CDKN3 shRNA cells compared with LACZ shRNA cells in one-way ANOVA; n = 10). Error bars show mean values ± SEM. (F) CDKN3 knockout cells fail to activate the spindle checkpoint in response to three mitotic poisons. (G) CDKN3 knockdown disrupts the SAC response to three spindle poisons. HeLa GFP-H2B/mCherry–α-tubulin cells were transfected with control and CDKN3 siRNAs, and 48 h later treated with inhibitors for 24 h. Cells were fixed and imaged to count nuclear fractions. n = 3 counts per siRNA per condition. P < 0.0001 ( t test). Error bars represent mean values ± SEM. (H) CDKN3 is essential for the spindle checkpoint in human primary brain stem cells. The SC-23 cells were challenged with 200 nm taxol 72 h after siRNA transfection. Mitotic arrest occurs in control cells (green arrows), and CDKN3 knockdown led to multinucleation (red arrows).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Functional Assay, Transfection, Negative Control, Positive Control, Knock-Out, Expressing, Western Blot, shRNA

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Spindle checkpoint failure in CDKN3 siRNA-treated cells is not caused by off-target RNAi effects. (A) Overexpression of siRNA-resistant GFP-CDKN3 rescues the SAC failure phenotype in cells transfected with CDKN3 siRNA. Phenotype rescue in CDKN3 knockdown cells transfected with siRNA-resistant GFP-CDKN3 is indicated by mitotic arrest in taxol (red). No phenotype rescue and minimal GFP fluorescence were observed in CDKN3 siRNA cells transfected with siRNA-sensitive GFP-CDKN3 construct (top). Transfection with either GFP-CDKN3 construct resulted in a similar level of GFP-CDKN3 expression in cells pretransfected with control siRNA (bottom two rows). (B) Quantification of the rescue experiment. Error bars indicate SEM. n = 3 experiments; P < 0.0001 in t test. (C) Validated siRNAs against CDKN3 recapitulate the SAC failure phenotype. (D) Quantification of the SAC failure phenotype induced by three CDKN3 siRNAs shown in C. n = 9, P < 0.0001 in one-way ANOVA. (E) Validation of CDKN3 knockdown by four separate siRNAs in quantitative Western blots. n = 6, P < 0.0001 in one-way ANOVA. Note the normal CDKN3 signal in cells transfected with MAD2 siRNA. (F) Cells transfected with four separate CDKN3 siRNAs express normal MAD2 level. The MAD2/actin ratio was quantified by Western blotting. n = 6, (not significant) n.s. in one-way ANOVA for each CDKN3 siRNA. Note the MAD2 knockdown (P = 0.0025) in cells transfected with positive control MAD2 siRNA. (G) Normal localization of endogenous MAD2 on prometaphase kinetochores in cells transfected with CDKN3 siRNAs. Note the lack of MAD2 signal in cells transfected with validated MAD2 siRNA. (H) Quantification of MAD2-positive prometaphase kinetochores in cells transfected with CDKN3 siRNAs and MAD2 siRNAs. P < 0.0001 in ANOVA, n = 50 200-µm nuclear cross sections per siRNA. (I) MAD2 overexpression does not rescue the SAC failure phenotype induced by CDKN3 siRNA. Note the similar nuclear morphology of cells transfected with CDKN3 siRNA alone compared with CDKN3 siRNA followed by MAD2 overexpression. (J) The SAC failure in CDKN3 siRNA cells is not rescued by ectopic overexpression of MAD2. n = 10; n.s. in t test. (K) Expression of Flag-MAD2 in cells transfected with the construct used in rescue experiments (I and J).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Over Expression, Transfection, Fluorescence, Construct, Expressing, Western Blot, Positive Control

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is essential for normal mitosis. (A) Inducible knockdown of CDKN3 and MAD2 leads to multinucleation (arrows, 200× magnification). (B) Quantification of multinucleation in CDKN3 and MAD2 knockout cells. P < 0.0001 for both CDKN3 and MAD2 siRNA (one-way ANOVA; n = 5). Error bars represent mean values ± SEM. (C) Knockdown of CDKN3 and MAD2 decreases mitotic index in unsynchronized cells as shown by flow cytometry. P < 0.001 in one-way ANOVA; n = 6. Error bars represent mean values ± SEM. (D) Representative time-lapse frames of cells dividing 72 h after transfection with negative control siRNA (A) and CDKN3 siRNA (B). Note the shortened time between NEB (black arrows) and anaphase (red arrows) upon CDKN3 knockdown. Time-lapse images were taken on an automated imaging system (Pathway 855; BD) in a controlled environment (5% CO 2 , 37°C) every 192 s (3.2 min) using laser autofocus and a 20× NA 0.75 objective lens (Olympus); only every other frame from relevant sequences is shown for simplicity. (E) Frequency distributions of anaphase times. Movies of individual cells in unsynchronized populations were followed manually frame-by-frame to detect NEB. 76 control cells and 137 siCDKN3-transfected cells were measured in three independent experiments. P < 0.0001 in t test. (F) Gallery of mitotic cells transfected with CDKN3 siRNA. Note the multipolar spindles, unattached chromosomes, multinucleation, and cleavage furrows cutting through partially decondensed chromosomes. (G) Quantification of abnormal mitoses in unsynchronized CDKN3 and MAD2 knockout cells. P < 0.0001 (one-way ANOVA; n = 5). Error bars represent mean values ± SEM.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Knock-Out, Flow Cytometry, Transfection, Negative Control, Imaging

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is a nucleolar protein during interphase and controls G1/S transition. (A) Endogenous CDKN3 localizes to interphase nuclei. Detergent extraction before fixation helps visualize the nucleolar fraction of endogenous CDKN3 (bottom). The signal is lost from cells transfected with CDKN3 siRNA (right), demonstrating antibody specificity. (B) Endogenous CDKN3 colocalizes with nucleolin. CDKN3 foci (green) are surrounded by diffuse nucleolin staining (red) in each nucleolus. (C) CDKN3 phosphatase activity is not required for subcellular targeting of GFP-CDKN3. HeLa cells were transfected with wild-type GFP-CDKN3 (top) and phosphatase-dead GFP-CDKN3 C79S point mutant (bottom). Nucleolar GFP-CDKN3 is visible in detergent-extracted nuclei. At least 100 nuclei were visualized per construct and condition. (D) Representative cell cycle profiles of cells transfected with control siRNA and CDKN3 siRNA. (E) Mildly increased S-phase fraction in HeLa cells transfected with CDKN3 siRNA. P < 0.01 in t test; n = 4. (F) CDKN3 is required for serum starvation–induced G1 arrest. Serum-starved HCT cells transfected with control and CDKN3 siRNAs were pulsed with EdU in serum-free medium to visualize cells that enter S phase (see micrographs on the right; red arrows indicate EdU-positive nuclei). P < 0.0001 in t test; n = 4.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Transfection, Staining, Activity Assay, Mutagenesis, Construct

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 localizes to centrosomes. (A) Endogenous CDKN3 colocalizes with pericentrin during mitosis. (B) Stably overexpressed DDK-CDKN3 associates with centrosomes. (C) A small fraction of endogenous CDKN3 localizes to the midzone in anaphase. (D) Stably overexpressed DDK-CDKN3 colocalizes with KIF20A on midbodies in telophase. (E) A fraction of endogenous CDKN3 remains at centrosomes in interphase. (F) Localization of stably overexpressed DDK-CDKN3 to interphase centrosomes.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Stable Transfection

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 is essential for centrosome maintenance. (A) Abnormal centrosome clusters in HeLa GFP-CENPA/GFP–γ-tub cells transfected with CDKN3 siRNA. Endogenous pericentrin was visualized by immunofluorescence. Arrows indicate multinucleated cells with supernumerary centrosomes. (B) Loss of CDKN3 causes generation of supernumerary centrosomes. P = 0.0073 in t test; n = 5.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Transfection, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: CDKN3 modulates progression through mitosis via regulation of CDC2 phosphorylation at Thr-161. (A) The CDKN3 interface is conserved in human CDK2 (Protein Database [PDB] accession no. 1B39 ) and CDC2 (PDB accession no. 3LFQ ) kinases (GDSEID/DYK motifs, blue). Activation loops (magenta) include putative CDKN3 target residue (pThr160/161; yellow). CDC25 target sites are green. Regions of full conservation are red; other regions are gray. (B) Endogenous CDKN3 colocalizes with endogenous CDC2 on centrosomes during mitosis. (C) Endogenous CDC2 pThr161 localizes to centrosomes and the mitotic spindle during cell division. Dephosphorylation of CDC2 pThr161 occurs in anaphase. HeLa cells were stained with antibody recognizing CDC2 pThr-161 , anti–α-tubulin antibody, and Hoechst 33342. (D) CDC pThr161 localizes to kinetochores in early mitosis. CDC2 pThr161 (red) colocalizes with the kinetochore marker GFP-CENPA (green) in prometaphase but not in anaphase. (E) CDC2 pThr161 is dephosphorylated at exit from mitosis. Cells were arrested in G2 through 24 h of exposure to RO3306 and washed to trigger mitotic entry. Decreasing cyclin B1 levels indicate cell cycle progression toward the mitotic exit. (F) Hyperphosphorylation of CDC2 pThr161 in HeLa cells transfected with CDKN3 siRNA. Total CDC2 and CDC2 pTyr15 levels are unaffected by CDKN3 siRNA. (G) Recombinant CDKN3 inactivates recombinant CDC2/cyclin B in an in vitro kinase assay in a dose-dependent manner. Increasing amounts of recombinant CDKN3 (0.5–5 µg) were incubated for 30 min in kinase buffer with a constant amount of active CDC2–cyclin B kinase complex, CDC2 substrate (histone H1), and radioactive [P 32 ]γ-ATP. CDC2-dependent H1 phosphorylation was detected by autoradiography. 150 mM olomoucine (a CDK kinase inhibitor) was used as a control. (H) Cells transfected with CDKN3 siRNA fail to dephosphorylate CDC2 pThr161 in early anaphase. Note the normal CDC2 pThr-161 metaphase signal in control and CDKN3 siRNA cells. The CDC2 pThr-161 signal persists in CDKN3 siRNA cells during anaphase.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Activation Assay, De-Phosphorylation Assay, Staining, Marker, Transfection, Recombinant, In Vitro, Kinase Assay, Incubation, Autoradiography

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: A proteome-wide phospho-mass spectrometry screen identifies downstream mitotic effectors of CDKN3. (A) Strategy to identify the CDKN3-CDC2 targets. (B) Mass spectrometry reveals hyperphosphorylation of CKβ pSer-209 upon CDKN3 knockdown. Two samples per siRNA were analyzed in duplicate runs (with a total of four LC-MS/MS experiments); representative images are shown. (C) Western blot verification of proteomic screen. Note the increased phosphorylation of endogenous CKβ upon CDKN3 knockdown, whereas the total endogenous CKβ protein level remains the same. (D) Endogenous CKβ pSer-209 localizes to centrosomes. (E) Endogenous CKβ pSer-209 localizes to centrosomes throughout mitosis and disappears from centrosomes in telophase. (F) Western blot verification of CKβ knockdown. (G) CKβ is essential for mitotic spindle checkpoint. HeLa cells were transfected with CK2β siRNAs and treated with 100 nm taxol (24 h) 2 d after transfection. (H) Quantification of SAC failure resulting from CKβ knockdown. P < 0.0001 in one-way ANOVA; n = at least 5 counts per siRNA. Error bars represent mean values ± SEM.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Transfection

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Loss of CDKN3 expression and elevated CDK activity in glioblastoma. (A) CDKN3 is ubiquitously expressed in human brain. (B) The CDKN3 protein is expressed in healthy brain (red arrows). CDKN3 expression is diminished (green arrows) in human GBM tumors. Images are shown at 100× magnification. (C) Immunohistochemistry quantification confirms loss of the CDKN3 protein in GBM. Percentages of CDKN3-positive cells in healthy brain cores and tumor specimens were compared by t test (P = 0.0003). Error bars represent mean values ± SEM. (D) Activation of CDKs in GBMs. Immunohistochemistry reveals increased CDK substrate phosphorylation in brain tumors compared with normal brain. (E) Quantification of increased CDK activity in GBMs. Percentages of phospho-CDK substrate–positive cells in healthy brain cores and tumors were compared by paired t test (P = 0.0133). Error bars represent mean values ± SEM. (F) Decreased expression of CDKN3 accompanied by CDK activation in an independent cohort of human GBMs. Note the increased phosphorylation of CDK substrates in all tumors. (G) Quantification of CDKN3 expression in healthy brain and GBM. CDKN3 and actin were quantified in at least three independent Westerns per specimen, and the ratios were compared with healthy brain (one-way ANOVA; n = 3 experiments for each tumor and 13 experiments for healthy brain). Yellow columns indicate tumors with significantly decreased total CDKN3 expression. Broken lines: 95% confidence interval. Error bars represent mean values ± SEM. (H) Quantification of the CDKN3 expression in 26 GBMs. 23% of tumors (6/26) showed significant loss of CDKN3 expression (one-way ANOVA; n = 3 experiments for each GBM and 13 experiments for healthy brain).

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: Expressing, Activity Assay, Immunohistochemistry, Activation Assay

Journal: The Journal of Cell Biology

Article Title: The tumor suppressor CDKN3 controls mitosis

doi: 10.1083/jcb.201205125

Figure Lengend Snippet: Model of sequential CDC2 dephosphorylation during mitosis. Phosphatases identified in this work are marked with asterisks. Dephosphorylation of Tyr-15 by CDC25 activates CDC2 at mitosis entry, whereas dephosphorylation of Thr-161 by CDKN3, PP2A, and other phosphatases guides CDC2 through late mitosis.

Article Snippet: The Myc-DDK–tagged CDKN3 construct was purchased from OriGene Technologies as the entry clone, verified by direct sequencing, and directly subcloned into pEGFP-C1 vector (Takara Bio Inc.) to generate the GFP fusion construct for immunofluorescence.

Techniques: De-Phosphorylation Assay

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 1 TRIM28 interacts with NLRP3. a Identification of TRIM28 as a potential NLRP3 interactor in LPS-stimulated mouse peritoneal macrophages (PMs) by mass spectrum primed with LPS. b Immunoblot analysis of TRIM28 expression in LPS-stimulated mouse PMs (n = 3 independent experiments). c Confocal microscopic analysis of the colocalization of NLRP3 (Red) and TRIM28 (Green) in unstimulated mouse embryonic fibroblasts (MEFs). Scale bar, 10 μm. d IP analysis of endogenous association between TRIM28 and NLRP3 in LPS-stimulated or LPS-primed and ATP-activated mouse PMs (n = 3 independent experiments). e Immunoprecipitation (IP) analysis of the association between TRIM28 and NLRP3, ASC or Caspase-1 in HEK293T cells transfected with the indicated plasmids. Similar results were obtained from three independent experiments.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Western Blot, Expressing, Immunoprecipitation, Transfection

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 2 No effects of TRIM28 on NLRP3 inflammasome priming. a, c Immunoblot analysis of p-IκBα and IκBα in LPS-stimulated mouse PMs from WT or Trim28CKO mice (a) or PMs transfected with Ctrl siRNA or Trim28 siRNA (c). b, d RT-PCR analysis of Ilb and Nlrp3 mRNA in LPS-stimulated mouse PMs from WT or Trim28CKO mice (b) or PMs transfected with Ctrl siRNA or Trim28 siRNA (d). All data are represented as mean ± SD in b, d. Similar results were obtained from five independent experiments (a, c) and three independent experiments (b, d).

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 3 TRIM28 promotes NLRP3 inflammasome activation. a, b ELISA analysis of IL-1β, TNF, and IL-6 in supernatants of mouse PMs from WT or Trim28CKO mice following priming with LPS for 7 h and subsequent stimulation with ATP, Nig, or poly(dA:dT) for 1 h (a two-tailed t test WT vs. Trim28CKO, *p = 0.011241, **p = 0.005688). c Immunoblot analysis of supernatants (SN) and cell lysates (CL) of mouse PMs from WT or Trim28CKO mice, following priming with LPS and subsequent stimulation with ATP (n = 3 independent experiments). d ELISA analysis of IL-1β in supernatants from mouse PMs transfected with control (Ctrl) siRNA or Trim28 siRNA for 48 h, followed by priming with LPS for 7 h and subsequent stimulation with ATP for 1 h (two- tailed t test Ctrl siRNA vs. Trim28 siRNA, **p = 0.005134). e ELISA analysis of serum levels of IL-1β, TNF, and IL-6 of WT or Trim28CKO mice after i.p. LPS injection (PBS, n = 3; LPS, n = 8 per group, two-tailed t test WT vs. Trim28CKO, ***p = 6 × 10−6). All data are represented as mean ± SD in a, b, d, e (*p < 0.05; **p < 0.01; ***p < 0.001). Similar results were obtained from three independent experiments.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Transfection, Control, Injection

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 5 TRIM28 inhibits K48-linked ubiquitination of NLRP3. a Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by IP with NLRP3 antibody. b Immunoblot analysis of lysates from HEK293T cells transfected with HA-Ub (WT), HA-tagged K48-linked ubiquitin (K48-Ub), or HA-tagged K63-linked ubiquitin (K63-Ub), Myc-NLRP3, and Flag-TRIM28, together with MG132 (10 μM) treatment for 4 h and followed by IP with Myc antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged ubiquitin (HA-Ub), Myc-NLRP3, and Flag-TRIM28 WT or C651A, together with MG132 (10 μM) treatment for 4 h, and followed by IP with Myc antibody. d Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, together with Flag-TRIM28 WT or C651A. Similar results were obtained from three independent experiments.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 4 TRIM28 inhibits NLRP3 protein degradation. a Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and increasing amount of Flag-TRIM28 plasmid. b Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, following stimulation with LPS. c Immunoblot analysis of lysates of mouse PMs transfected with Ctrl siRNA or Trim28 siRNA for 48 h, following stimulation with LPS. d, e Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h and then treated cycloheximide (CHX) for the indicated time periods. NLRP3 expression was quantitated by measuring band intensities using the “ImageJ” software. The values were normalized to Actin. Data are represented as mean ± SD (two-tailed t test WT vs. Trim28CKO, ***p = 1 × 10−12, 3 × 10−17). f Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or MG132 (10 μM) treatment for 4 h. g Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or chloroquine (50 μM) treatment for 4 h. Similar results were obtained from three independent experiments.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Software, Two Tailed Test

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 6 TRIM28 promotes SUMOylation of NLRP3. a, b Immunoblot analysis of lysates of mouse peritoneal macrophages from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by SUMOylation assays with NLRP3 antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and stimulated with an increasing amount of 2-D08 for 24 h. d–f Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, His-UBC9 and Flag-TRIM28 WT or C651A, and HA-SUMO1, HA-SUMO2, or HA-SUMO3, followed by MG132 (10 μM) treatment for 4 h, and then performed SUMOylation assays with Myc antibody. Similar results were obtained from three independent experiments.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Western Blot, Transfection

Journal: Nature communications

Article Title: TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation.

doi: 10.1038/s41467-021-25033-4

Figure Lengend Snippet: Fig. 7 Working model for TRIM28 promoting NLRP3 inflammasome activation. TRIM28 binds to NLRP3, promotes SUMOylation of NLRP3, and thus inhibits NLRP3 ubiquitination and degradation in proteasome, resulting in the enhancement of NLRP3 inflammasome activation.

Article Snippet: TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added.

Techniques: Activation Assay, Ubiquitin Proteomics

Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Effect of TRIM24, TRIM33 and TRIM5α B boxes on L1 retrotransposition in HeLa cells. ( A ) Amino acid sequence alignment of TRIM24, TRIM33, TRIM5α and TRIM28 B boxes (24BB, 33BB, 5αB2 and 28BB, respectively) is performed using Clustal Omega method. Stars indicate the positions of the three amino acids where TRIM28 B box mutations are introduced (shown in Figure ). ( B ) Western blot analysis of the proteins expressed by B box constructs described in A. All indicated B box constructs are FLAG tagged on the C terminus to allow their detection. GAPDH is used as loading control. ( C ) Results of L1 retrotransposition assay in HeLa cells co-transfected with L1Neo-expression plasmid and one of the plasmids containing constructs shown in A. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t -test, **** P < 0.0001, ## P = 0.0016) Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Sequencing, Western Blot, Construct, Control, Transfection, Expressing, Plasmid Preparation, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Both human TRIM28 (H-TRIM28) and mouse TRIM28 (M-TRIM28) increase L1-Neo retrotransposition. ( A ) Grey bars represent results of L1 retrotransposition assay in HeLa cells co-transfected with a plasmid expressing a Neo-tagged, full-length human wild type L1 and either an empty plasmid (control) or a plasmid expressing either H-TRIM28 or M-TRIM28. Images of flasks containing Neo-resistant (Neo R) colonies corresponding to L1Neo retrotransposition are shown above the graph. Purple bars are results of toxicity assay for which the pIRES2-EGFP vector carrying a constitutive Neo-resistant expression cassette (Neo R) is co-transfected with the control, H-TRIM28, or M-TRIM28 expressing plasmids. ( B ) Western blot analysis of endogenous TRIM28 expression in wild type U2OS cells (U2OS WT) and U2OS TRIM28 knock-out cells (U2OS KO). U2OS KO and U2OS WT cells transfected with a plasmid expressing FLAG tagged human TRIM28 are used as positive control. The lower molecular weight band (indicated by the arrow) corresponds to TRIM28. GAPDH is used as loading control. ( C ) L1 retrotransposition and toxicity assays performed in U2OS WT cells using plasmids and conditions described in (A). ( D ) L1 retrotransposition and toxicity assays performed in U2OS KO cells using plasmids and conditions described in (A). For all experiments asterisks (*) denote statistical significance between indicated experimental data points and the control ( n = 3, t -test, *** P < 0.001, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Knock-Out, Positive Control, Molecular Weight, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Human and Mouse TRIM28 specifically interact with human L1 ORF2 protein. ( A1 ) Schematic of a full length L1, containing 5’UTR, two open reading frames (ORF1 and ORF2) and 3’UTR ending with a polyA site and a polyA tail. ( A2 ) Schematic of plasmids used for co-Immunoprecipitation assay in HeLa cells. ORF1p is not tagged. T7 indicates the position of the T7 tag in the ORF2p expressing plasmid. FLAG indicates the position of the FLAG tag in the TRIM28 expressing plasmid. ( B ) Results of co-IP using lysates of HeLa cells transfected with indicated plasmids (+) and anti-FLAG-beads assessed by western blot analysis. ORF1 (the upper bands slightly above GAPDH) is detected using anti-ORF1 antibodies. TRIM28 is detected using anti-FLAG antibodies. GAPDH is used as loading control (the lower bands). Input corresponds to assessment of protein expression in whole cell lysates. CoIP corresponds to the assessment of co-IP results. ( C ) Results of co-IP using lysates of HeLa cells transfected with indicated plasmids (+) and anti-FLAG-beads assessed by western blot analysis. ORF2p is detected using anti-T7 antibodies. TRIM28 is detected using anti-FLAG antibodies. GAPDH is used as loading control. The arrow indicates a non-specific band in the input lysates that masks detection of transfected ORF2p in HeLa cells. The asterisk indicates an ORF2p-specific band.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Co-Immunoprecipitation Assay, Expressing, Plasmid Preparation, FLAG-tag, Transfection, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: N-terminal B box containing TRIM28 fragments increase L1 retrotransposition. ( A ) Schematic of TRIM28 fragments tested in the L1 retrotransposition assay. All fragments are generated from Human TRIM28, and FLAG-tagged at the C terminus. Names of constructs are reported on the left. The amino acid coordinates corresponding to each fragment are described in materials and method. ( B ) Results of L1 retrotransposition assay in HeLa cells using plasmids depicted in A. The number of Neo R colonies resulting from co-transfection of an empty plasmid with a plasmid expressing Neo-tagged, full-length human wild type L1 is used as control (control). Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t -test, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Generated, Construct, Cotransfection, Plasmid Preparation, Expressing, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Amino acids involved in TRIM28 multimerization are required for its ability to increase L1 retrotransposition. ( A ) Schematic of TRIM28 B box variants. BB is B box, WT is wild type, single or triple mutations are indicated using single letter amino acid code and amino acid position in the human wt TRIM28 protein. ( B ) L1 retrotransposition result. HeLa cells are transiently co-transfected with plasmids expressing human neomycin tagged L1 (L1Neo) and indicated TRIM28 BB variants. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. ( C ) Schematic of full-length wild type TRIM28 (TRIM28 WT) and triple mutant TRIM28 (TRIM28 3M). Amino acid mutations are noted as described in A. ( D ) L1 retrotransposition result. HeLa cells are transiently co-transfected with plasmids expressing human neomycin tagged L1 (L1Neo) and indicated full-length TRIM28 variants. Images of flasks containing Neo R colonies corresponding to L1Neo retrotransposition are shown above the graph. Asterisks (*) denote statistical significance between listed constructs and the control ( n = 3, t-test, **** P < 0.0001). Dots represent number of Neo R colonies observed in individual experiments. Error bars represent the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Transfection, Expressing, Mutagenesis, Construct, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Results of analysis of differentially expressed genes in HeLa cells over-expressing control plasmid, TRIM28 WT, or TRIM28 3M. ( A ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 WT versus Control. The horizontal grey line indicates P = 0.05. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. Multiple DNA repair genes are significantly downregulated in TRIM28 WT, compared to Control. ( B ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 WT versus TRIM28 3M. The horizontal grey line indicates P = 0.05. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. Multiple DNA repair genes are significantly downregulated in TRIM28 WT, compared to TRIM28 3M. ( C ) The volcano plot shows differentially expressed genes in HeLa cells overexpressing TRIM28 3M versus Control. The horizontal grey line indicates P = 0.05 in Wald test. A fold change cutoff of 1.1 in the graph is shown as the dashed lines running parallel to the y-axis. The indicated DNA repair genes are not differentially expressed in TRIM28 3M compared to Control. ( D ) Heatmap of normalized expression of individual DNA repair genes that are significantly differentially expressed in HeLa cells transfected with TRIM28 WT compared to the control and TRIM28 3M expression plasmids. (Wald test, P < 0.05).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Expressing, Control, Plasmid Preparation, Transfection

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of cDNA products generated by the ORF2p in HeLa cells transfected with wild-type or mutant TRIM28 or TRIM28 B Box. ( A ) Flow chart of the LEAP assay adapted from ( , ). ORF2p-generated cDNA is detected by PCR with a step wise set of ORF2 sequence specific forward primers (O1-O4) and a reverse primer Ro. In parallel, conventional RT-PCR was performed with the same set of step wise ORF2 primers. O1-O4: forward ORF2 specific primers. The expected length of PCR products is shown on the right. ( B , C ) LEAP samples are prepared by harvesting HeLa cells 48h post-transfection with indicated constructs and analyzed with indicated sets of primers. Control is LEAP prep on cells transfected with the empty plasmid (i.e. no ORF2p expression). RNA integrity in LEAP preps is assessed with the same set of ORF2 specific primers shown in A. A PCR product expected to be produced with O4 primer is absent in cells expressing WT full-length H-TRIM28 (TRIM28 WT) or WT B box (BB WT). Mutations of three amino acids responsible for multimerization (TRIM28 3M) eliminate this effect.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Generated, Transfection, Mutagenesis, Sequencing, Reverse Transcription Polymerase Chain Reaction, Construct, Control, Plasmid Preparation, Expressing, Produced

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of L1 insertion in genomic DNA using ruler PCR assays . ( A ) Schematic of the ruler PCR assay adapted from . Three kilobase ruler PCR was performed using primers covering the 3kb target band. The position of primers relative to the L1 vector are shown. ( B ) PCR on genomic DNA sequence of HeLa cells transfected with L1-neo constructs was performed. Forward primer (F) and reverse primer (R) are applied. The L1-neo plasmid gives a band at 3771 bp, while the spliced L1-neo insertion gives a band at 2864 bp. Thirty-two clones were randomly picked and subjected to genomic DNA extraction. Left, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and the control plasmid (PCDNA 3.1 empty vector). Middle, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and TRIM28 WT plasmid. Right, number of clones with or without the spliced 3kb L1-Neo insertion in HeLa cells co-expressing L1-Neo construct and TRIM28 3M plasmid.

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Plasmid Preparation, Sequencing, Transfection, Construct, Clone Assay, DNA Extraction, Expressing, Control

Journal: Nucleic Acids Research

Article Title: A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

doi: 10.1093/nar/gkad247

Figure Lengend Snippet: Analysis of length of tumor specific L1 insertions in WGS data set collected from endometrial, prostate, and ovarian cancer patients. ( A ) Sixteen patients with endometrial cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( B ) Length of tumor specific de novo L1 inserts in endometrial cancer patients is significantly shorter in high TRIM28 group ( n = 324) than low TRIM28 group ( n = 491), t -test, P < 0.0001. ( C ) Sixteen patients with prostate cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( D ) Length of tumor specific de novo L1 inserts in prostate cancer patients is significantly shorter in high TRIM28 group ( n = 293) than low TRIM28 group ( n = 326), t -test, P = 0.0135. ( E ) Sixteen patients with ovarian cancer are selected and grouped into two groups ( n = 8) according to high or low TRIM28 mRNA expression levels ( t -test, P < 0.0001). ( F ) Length of tumor specific de novo L1 inserts in ovarian cancer patients is significantly shorter in high TRIM28 group ( n = 383) than low TRIM28 group ( n = 386), t -test, P = 0.0319. For each individual figure, error bar represents the standard deviation (SD).

Article Snippet: Plasmids containing DNA encoding human and mouse TRIM28 were obtained from Origene (RC201205 and MR210883, respectively) and subcloned into a PCDNA 3.1/Hygro+ vector using NheI and HindIII restriction enzymes.

Techniques: Expressing, Standard Deviation