mouse trim28  (OriGene)

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Skeletal phenotypes of WT and Trim28 MKO mice at E19.5 visualized with Alcian blue and Alizarin red staining; arrowheads indicate thicker and shorter ribs and over-expanded hindlimb cartilage. Scale bar, 0.5 cm. (B) E17.5 mouse proximal tibial sections stained with Alcian blue Hematoxylin/Orange G. Gross image (left) and magnified views of each zone (right) are shown(RZ, resting zone; PZ, proliferating zone; HZ, hypertrophic zone). Scale bar, 200 μm. (C) Increased cell proliferation in the Trim 28 MKO proximal tibial sections (E17.5) as indicated by Ki67 immunohistochemistry (IHC). Bottom panel: area boxed in red is the magnified perichondrium (PC), while the yellow boxed area is the magnified periarticular resting zone (PRZ). Scale bars, 200 μm. (D) Type II collagen IHC of proximal tibial sections (E17.5). Area boxed in red is magnified in the bottom panels showing high levels of type II collagen retention inthe Trim28 MKO HZ. Scale bars, 200 μm. (E) Type X collagen IHC staining of distal femoral sections (E17.5). The red box area is magnified in the bottom panel, and red dotted lines define type X collagen-expressing areas. Scale bars, 200 μm. See also .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Distribution of 13,683 qualified single cells from WT (6,235) and Trim28 MKO (7,448) hindlimb GPs in 8 clusters were visualized by uniform manifold approximation and projection (UMAP). (B) UMAP in (A) is divided into two genotype-specific UMAPs. (C) The proportion of each cell cluster in total WT or Trim28 MKO hindlimb GPs. (D) Enriched Wikipathways and KEGG pathways in Trim28 MKO -U cluster versus all cells, which includes total WT and Trim28 MKO cells. (E) Violin plots of the expression of previously reported mesenchymal progenitor/SSC marker genes in each of the eight cell clusters identified in (A). (F) Distribution of SSCs in scRNA-seq UMAPs (the same as B). The criteria for defining SSCs here are cells positive for Itgav and Cd200 and negative for Ptprc , Tek , Thy1 , Enpep , and Eng in mRNA expression. Dashed black line indicates the boundary of the Trim28 MKO -U cluster. (G) Colony-formation assay initiated by seeding 1,000 (top panels) or 100 (bottom panels) rib GP cells. Clones containing at least 50 cells from the 100-cell seeding group are quantified in the bottom (n = 6). p value = 0.0081. Scale bar, 1 cm. Comparisons are conducted by Student’s t test, two tailed. Data are presented as mean ± SEM. (H) Representative flow cytometric analysis of the proportion of SSCs (CD45 − Ter119 − TIE2 CD51 + 6C3 − THY1.2 − CD105 − CD200 + ) and pre-BCSPs (CD45 − Ter119 − TIE2 − CD51 + 6C3 THY1.2 − CD105 − CD200 − ) in hindlimb GPs (n = 3 for each genotype). The representative flow image in the bottom shows the Trim28 MKO sample has a population shift from pre-BCSP to SSC when gated by CD200 expression. p values are shown in the figure. All comparisons are conducted by Student’s t test, two tailed. Data are presented as mean ± SEM. and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Expressing, Marker, Colony Assay, Clone Assay, Two Tailed Test

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Dot plots comparing the expression of selected lineage/niche markers in total WT SSCs versus Trim28 MKO SSCs. (B) Ex vivo chondrogenesis (top panels) and osteogenesis (bottom panels) of FACS WT or Trim28 MKO GP SSCs. Representative staining is shown (n = 3 per genotype). Scale bar, 400 μm. (C) UMAP of 5 subclusters of SSCs from combined WT and Trim28 MKO hindlimb GP cells (top panel). Genotype-specific UMAPs of SSCs are shown in the bottom panels. (D) RNA velocity trajectory inference analysis of gross SSCs (left). The separated WT (top panel) and Trim28 MKO (bottom panel) velocity trajectories are shown on the right. (E) Proportion of each SSC subtype in WT or Trim28 MKO GP SSCs. (F) Enriched WiKiPathways and MSigDB_Hallmarks in Trim28 MKO -NSSCs versus all SSCs (WT plus Trim28 MKO ). (G) Dot plots comparing the expression of osteochondrogenic marker genes in WT and Trim28 MKO root-SSCs, Trim28 MKO -NSSCs, WT chondro- and osteo-SSCs. (H) Transcriptomic profiles of Trim28 MKO SSCs (MKO), but not WT SSCs (WT), show a strong similarity with neural crest cells (NCCs) according to Spearman correlation analysis. (I) Pseudo-bulk-level transcriptomic similarity analysis of NCC and SSC subtypes. The size of dots represents the cell number of each subcluster. (J) FACS Trim28 MKO GP SSCs exhibit more potent neurogenic differentiation than WT SSCs. Representative bright-field images are shown in the left panels, and percentages of neuron-like cell/total cell and neurite length/neuron-like cell are quantified in the right panel (n = 4 per genotype). Scale bar, 100 μm. p < 0.0001. Data comparisons are conducted by Student’s t test, two-tailed. Data are presented as mean ± SEM. (K) Trim28 MKO SSCs express higher levels of neurogenic markers than WT SSCs after 5 days of neurogenic induction (n = 3 per genotype). p values are shown in the figure. Data comparisons are conducted by Student’s t test, two-tailed. Data are presented as mean ± SEM. See also and and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Expressing, Ex Vivo, Staining, Marker, Two Tailed Test

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Volcano plot of RNA-seq data demonstrating significant changes in gene expression between WT and Trim28 MKO rib GP cells. Top 10 up- and down-regulated genes are shown. (B) mTORC1 and PI3K-AKT pathways are enriched in Trim28 MKO samples by gene set enrichment analysis (GSEA). (C) Global changes of H3K9me3 (left panel) and DNA methylation (right panel) caused by Trim28 deletion. (D) Trim28 MKO GP cells have decreased global H3K9me3 modifications at gene bodies (left panel) and enhancers (right panel). (E) Regions with down-regulated H3K9me3 (upper panel) and CpG DNA methylation (lower panel) show enriched binding motifs of TFs related to pluripotency and neurogenesis. (F) Chromatin accessibility status of SSCs and NCCs at the genomic regions that are silenced by TRIM28 through H3K9me3 (left panel) and CpG DNA methylation (right panel). (G) Proportion of TRIM28-silenced regions that contain TEs. The chromatin accessibility information is from (F). (H) Venn diagram of genes with upregulated mRNA expression (mRNA up), genes silenced by TRIM28 through H3K9me3 (H3K9me3 down), and genes silenced by TRIM28 through CpG DNA methylation (CpG DNA methylation down) identified 124 TRIM28-silenced genes. (I) DAVID tissue expression analysis of the 124 TRIM28-silenced genes. (J) DAVID GO biology process analysis of the 124 TRIM28-silenced genes. (K) TRIM28 silences the gamma-protocadherin gene cluster through H3K9me3. See also , , , , and .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: RNA Sequencing, Gene Expression, DNA Methylation Assay, Binding Assay, Expressing

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) pS6 (an mTORC1 downstream effector) and pAKT (an mTORC1 upstream activator) levels are increased in E17.5 Trim28 MKO rib GPs (WT, n = 4; Trim28 MKO , n = 5). (B) pS6 level is increased in E17.5 Trim28 MKO mouse distal femur. GP areas are demarcated by white dashed lines. Red boxes are magnified in the bottom panel. Scale bars, 200 μm. (C) GREM1 and TRIM28 protein expressions are inversely related in the rib GP cells (WT, n = 3; Trim28 MKO , n = 2). (D) mTORC1 and AKT pathways are activated by recombinant GREM1 in ATDC5 cells in a time-dependent manner. (E) Levels of pAKT and pS6 were reduced by Grem1 gene knockdown with shRNA targeting Grem1 (sh Grem1) in cultured Trim28 MKO rib GP cells compared with control shRNA (shct). Densitometry quantification (n = 4) is shown on the right. p values are shown in the figure. Comparisons are conducted using one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. (F) H3K27ac and H3K9me3 marks at the promoter region of the Grem1 gene. (G) The occupancy status of CpG DNA methylation and H3K27ac at DMR1 and DMR2. The most distinctive changes are highlighted, and DMR regions are indicated with dark blue bars. (H) Luciferase reporter assays indicating the transcriptional activity of the Grem1 enhancers (DMR1 or DMR2) and promoter in ATDC5 cells. Schematics of the reporter constructs are in the top panel. Empty pGL3-basic vector was used as a negative control. Representative quantified results are shown in the bottom panel (n = 6). p values are shown in the figure. Comparisons are achieved using Student’s t test, two-tailed. Data are presented as mean ± SEM. (I) Grem1 mRNA levels in control (sg GFP ) and CRISPR-Cas9-mediated DMR1 (sgDMR1) or DMR2 (sgDMR2–1, sgDMR2–2) deleted WT and Trim28 MKO rib GP cells (n = 6). p values are shown in the figure. Comparisons are conducted using one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. See also .

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Recombinant, Knockdown, shRNA, Cell Culture, Control, DNA Methylation Assay, Luciferase, Activity Assay, Construct, Plasmid Preparation, Negative Control, Two Tailed Test, CRISPR

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet: (A) Skeletal phenotypes of the E17.5 vector-, YL294002-, or rapamycin-treated mice visualized with Alcian blue and Alizarin red staining. Red arrows indicate reduced knee cartilage and black arrows indicate expanded rib cages in YL294002-and rapamycin-treated mice. Scale bars, 0.5 cm. (B) Representative Alcian blue Hematoxylin/Orange G stain of proximal tibial (right) and distal femoral (left) sections from mice treated with vector, YL294002, and rapamycin. Red arrows indicate rescued proliferating and hypertrophic zones. Scale bars, 200 μm. (C) Ex vivo neurogenesis of GP cells isolated from WT hindlimb treated with either GREM1 (300 ng/mL) or PBS in either neurogenic media or αMEM (n = 5 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. Scale bars, 100 μm. (D) Expression of key neurogenic markers (n = 3 per group) in cells cultured as described in (C). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are mean ± SEM. (E) Rapamycin (100 nM) treatment partially rescues the enhanced neurogenesis of hindlimb GP cells caused by Trim28 loss. DMSO was used as a negative control (n = 5 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM. Scale bars, 100 μm. (F) mRNA expression of key neurogenic markers from cells treated as described in (E) (n = 3 per group). p values are shown in the figure. Comparisons are achieved by one-way ANOVA analyses with Tukey’s post-hoc test. Data are presented as mean ± SEM.

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Plasmid Preparation, Staining, Ex Vivo, Isolation, Expressing, Cell Culture, Negative Control

Journal: Cell reports

Article Title: TRIM28 secures skeletal stem cell fate during skeletogenesis by silencing neural gene expression and repressing GREM1/AKT/mTOR signaling axis

doi: 10.1016/j.celrep.2023.112012

Figure Lengend Snippet:

Article Snippet: Mouse: Trim28 f/f , Jackson Laboratories , Cat # 018552.

Techniques: Virus, Recombinant, Plasmid Preparation, SYBR Green Assay, cDNA Synthesis, DNA HS Assay, Reporter Assay, Software, Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 plays a mechanistic role in tumor progression by recruiting MDSCs into the tumor microenvironment. ( A ) TRIM28 expression levels in different tumor types from TCGA database were analyzed by TIMER2.0 (* p < 0.05, ** p < 0.01, *** p < 0.001). ( B ) Survival analysis comparing the high and low expression of TRIM28 in lung adenocarcinoma according to TCGA dataset by using the website GEPIA 2 ( http://gepia2.cancer-pku.cn/#survival ). The high or low expression of TRIM28 were divided according to 50% of the total sample. ( C ) Immunohistochemical analysis of TRIM28 protein levels in NSCLC samples on tissue microarrays. Representative examples of TRIM28 expression in adjacent non-cancerous lung tissues, NSCLC tissues are shown. The scale bars represent 100 μm. ( D ) Overall survival analysis of patients with NSCLC stratified by the TRIM28 expression level in 90 samples. Kaplan-Meier survival analysis indicating a significant association between higher TRIM28 expression and poorer OS in NSCLC. ( E ) The correlations of TRIM28 expression and MDSCs infiltration in pan-cancers were analyzed by TIMER2.0. ( F ) Correlation of TRIM28 expression, tumor purity, and MDSCs infiltration in TCGA lung adenocarcinoma (LUAD) and lung squamous cell cancer (LUSC). The expression of TRIM28 positively correlates with MDSCs infiltration in NSCLC. ( G - H ) Representative immunofluorescence staining of CD14 and TRIM28 in tissue from human lung adenocarcinoma and the correlation between TRIM28 and CD14 intensity. The expressions of TRIM28 and CD14 were measured with mean fluorescence intensities (MFIs) (in arbitrary units, a.u.), respectively. The pearson correlation between TRIM28 and CD14 expression (n = 90; p < 0.01, r = 0.567). Scale bars: 50 μm. ( I - J ) Cox regression analyses using data from TCGA indicated that high MDSC infiltration was significantly associated with poorer prognosis in NSCLC. Furthermore, elevated TRIM28 expression and a high proportion of MDSCs significantly correlated to poorer OS compared to their counterparts, strongly suggesting that TRIM28 influenced patient prognosis through an immune-related mechanism. Split infiltration percentage of patients: 50% ( I ). Split expression percentage of patients: 50% and split infiltration percentage of patients: 50% ( J )

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 inhibition enhances anti-PD-1 therapy in a syngeneic lung cancer model. ( A ) The schematic of tumor inoculation and treatment in mice. Western blot validated TRIM28 knockout or overexpression in CMT-167 cells. ( B ) C57BL/6J mice were subcutaneously injected with shControl or shTRIM28 CMT-167 cells and weekly with anti-PD-1 (200 µg/mouse) or isotype control (200 µg/mouse) by intraperitoneal injections starting on day 7 post tumor cell injection. Tumor growth curves and tumor weight were shown. n = 5 mice per treatment group. * p < 0.05; ** p < 0.01. ( C ) C57BL/6J mice were subcutaneously injected with Control or TRIM28 CMT-167 cells and weekly with anti-PD-1 (200 µg/mouse) or isotype control (200 µg/mouse) starting on day 7 post-tumor cell injection. Tumor growth curves and tumor weight were shown. n = 5 mice per treatment group. * p < 0.05; ** p < 0.01. ( D ) Experimental strategy. ( E ) Humanized huHSC-NOG-EXL mice were injected with shControl or shTRIM28 H1299 cells and treated with anti-PD-1(200 µg/mouse). Control animals were treated with isotype control. Tumor growth curves and tumor weight were shown. The treatment schema is as in ( D ). n = 5 mice per treatment group. Statistics were calculated using a one-way ANOVA post hoc Tukey test. * p < 0.05; ** p < 0.01

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Inhibition, Western Blot, Knock-Out, Over Expression, Injection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 is required for K63-linked ubiquitination of RIPK1. ( A ) Ectopic HA-TRIM28 interacts with Flag-RIPK1 in 293T cells. Endogenous TRIM28 interacts with endogenous RIPK1 in CMT-167 and H1299 cells. ( B ) HEK293 cells were transfected with HA-TRIM28 and Flag-RIPK1 as indicated. Cell lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-ubiquitin, anti-RIPK1, or anti-HA as indicated. ( C and D ) RIPK1 ubiquitination is increased upon overexpression of TRIM28-WT but not the ΔR mutant. 293T cells were transfected with HA-TRIM28 WT or ΔR mutant, and the cell lysates were subjected to immunoprecipitation using anti-Flag antibodies ( C ) or Ni-NTA pull-down under denaturing conditions ( D ), followed by immunoblotting with the indicated antibodies ( E ) RIPK1 ubiquitination was decreased upon TRIM28 depletion. 293T cells were co-transfected with the indicated plasmids or shRNAs, and Flag-RIPK1 was immunoprecipitated and analyzed by immunoblotting. ( F ) H1299 cells were transfected with control or TRIM28 shRNA, and endogenous RIPK1 was immunoprecipitated and analyzed for ubiquitination ( G ) TRIM28 modified RIPK1 by K63-linked ubiquitination. Cell lysates prepared in ( C ) were immunoprecipitated with anti-Flag and blotted with anti-ubiquitin K63 and anti-ubiquitin K48

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Transfection, Immunoprecipitation, Over Expression, Mutagenesis, Western Blot, shRNA, Modification

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 activates NF-κB signaling. ( A ) HEK293 cells were co-transfected with the indicated plasmids along with pNF-κB-luc plasmids or the control-luciferase plasmid and subjected to a reporter assay. The luciferase assay showed that TRIM28, but not the ΔR mutant, induced the activation of NF-κB signaling. n.s., not significant; ** p < 0.01. ( B ) p-IκBα, IκBα, p-IKKα/β, IKKα, IKKβ, p-p65, p65, TIRM28, and β-actin detected by western blot in TRIM28-knockdown and TRIM28-overexpressed CMT-167 cells. ( C ) p-IκBα, IκBα, p-IKKα/β, IKKα, IKKβ, p-p65, p65, TIRM28, and β-actin detected by western blot in TRIM28-knockdown and TRIM28-overexpressed H1299 cells. ( D ) Western blotting analysis of IκBα expression in the indicated cells treated with TNF-α (10ng/ml). β-actin is used as a loading control. ( E ) Assay of NF-κB luciferase reporter gene activity in TRIM28-overexpressing CMT-167 and H1299 cells transfected with vector or the IκBα dominant negative mutant (IκBα-mu). ** p < 0.01. In ( A ) to ( C ), and ( E ), analyses were done in triplicate. Data represent mean ± SEM from each of three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Transfection, Luciferase, Plasmid Preparation, Reporter Assay, Mutagenesis, Activation Assay, Western Blot, Expressing, Activity Assay, Dominant Negative Mutation, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 induces CXCL1 via the NF-κB pathway. ( A - B ) Expression of p65 was determined by western blot in CMT-167 or H1299 cells overexpressing wild-type TRIM28 or TRIM28 knockdown with shRNA. The whole cell lysates and nuclear protein extracts were subjected to immunoblotting analysis. Data are representative of results obtained in three independent experiments. ( C ) The immunofluorescence assays of p-65 were performed in CMT-167 cells treated with the indicated plasmids. DAPI (blue) was used as a nuclear counterstain. The quantification of nuclear p-65 positive staining in at least 200 counted cells was presented as percentage ± SEM. Statistics calculated using one-way ANOVA post hoc Tukey for multiple comparisons. ( D - E ) RT-qPCR determination of CXCL1 mRNA expression in TRIM28-knockdown and TRIM28-overexpressed CMT-167 or H1299 cells, treated with or without NF-κB inhibitor (BAY11-7085) at 10µM for 24 h. ELISA validation of levels of CXCL1 in cell culture supernatants from CMT-167 or H1299 cell culture. Data are representative of results obtained in three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. ( F - G ) Representative IHC staining and quantification for CXCL1 in the indicated tumor models are shown. Additionally, ELISA was performed to validate the levels of CXCL1 in tumor lysates and sera from CMT-167 syngeneic tumor models, including subcutaneous tumors and blood samples collected from mice bearing CMT-167 control and CMT-167-shTRIM28 tumors. The scale bar represents 50 μm. ( H - I ) Representative IHC staining and quantification for CXCL1 in the indicated tumor models are displayed. Additionally, ELISA was conducted to validate the levels of CXCL1 in tumor lysates and sera from CMT-167 syngeneic tumor models, including subcutaneous tumors and blood samples collected from mice bearing CMT-167 control and CMT-167-TRIM28 tumors. The scale bar represents 50 μm. Statistical analysis was performed using a two-tailed student’s t-test. ** p < 0.01

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Western Blot, shRNA, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: TRIM28 Promotes MDSCs recruitment in autochthonous tumors from KP mice. ( A ) Migration of MDSCs toward conditioned medium (CM) from CMT-167 cells were transfected with the indicated plasmids, treated with or without immunoglobulin G (IgG) control or CXCL1-neutralizing antibody. Data are representative of results obtained in three independent experiments. ( B ) Correlation of CXCL1 expression, tumor purity, and MDSCs infiltration in TCGA lung adenocarcinoma and lung squamous cell cancer. The expression of CXCL1 positively correlates with MDSCs infiltration in NSCLC. ( C ) Schematic representation of lentiviral TRIM28 or GFP overexpression in KP mice. KP mice were anesthetized with sodium pentobarbital followed by intranasal injection of Ad-Cre (1.5 × 10 6 pfu/mouse). ( D ) Representative images of HE staining in tumor-burdened lungs of KP mice were analyzed. ( E - F ) Representative IHC ( E ) and IHC quantification ( F ) for immune cell markers (CD8, CD8 + T cells; Gr-1, S100A8, S100A9, MDSC cells) in indicated in indicated tumor models. The scale bar represents 50 μm. Statistics calculated using a one-way ANOVA post hoc Tukey test. ( G ) Kaplan-Meier survival analysis of KP mice infected with Lenti-GFP-Cre or Lenti-TRIM28-Cre. Log-rank test. ( H - I ) Flow cytometry gating strategy of immune cells. Quantification of flow-cytometry data for CD8 + T, CD4 + T, MDSCs as a percentage of leukocytes (CD45 + ) in tumor-burdened lungs from indicated tumor models. ( J ) Real-time qPCR and western blotting showed expression of representative MDSCs immunosuppressive gene in the indicated cell lines. Data are representative of results obtained in three independent experiments. ( K ) Quantification of the proliferation of CFSE-labelled CD8 + T cells cocultured with MDSCs from tumor-burdened lungs of KP or KP-TRIM28 mice, analyzed by flow cytometry. Data are representative of results obtained in three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. ** p < 0.01

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Migration, Transfection, Expressing, Over Expression, Injection, Staining, Infection, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: E3 ligase TRIM28 promotes anti-PD-1 resistance in non-small cell lung cancer by enhancing the recruitment of myeloid-derived suppressor cells

doi: 10.1186/s13046-023-02862-3

Figure Lengend Snippet: Targeting RIPK1 increases the sensitivity of lung tumors to anti-PD-1 therapy. ( A ) Kaplan-Meier curves predicting survival of LUSC patients receiving anti-PD-1 therapy based on net changes in TRIM28 mRNA levels in the LUSC-GSE93157-anti-PD-1 datasets. ( B ) Kaplan-Meier curves predicting survival of melanoma patients receiving anti-PD-1 therapy based on net changes in TRIM28 mRNA levels in the melanoma-GSE91061-anti-PD-1 datasets. ( C - D ) C57BL/6J mice were subcutaneously injected with CMT-167 cells and treated with anti-PD-1, PK68 (RIPK1 inhibitor), PK68 plus anti-PD-1, or isotype control and vehicle. Tumor growth was monitored until the experimental endpoints. Data are shown as mean ± SEM. Tumor growth curves were shown. ( E - F ) Representative images of IHC for CD8, Gr-1, and S100A8 + S100A9 in indicated mouse tumors ( E ) and IHC quantification ( F ). The scale bars represent 50 μm. Error bars indicate mean ± SEM. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons. ** p < 0.01. ( G ) High TRIM28 expression is positively correlated with MDSCs infiltration in multiple cohorts of cancer patients. TRIM28 promotes NF-κB activation by regulating K63-linked ubiquitination of RIPK1, leading to increased expression of the cytokine CXCL1, a chemoattractant for MDSCs via the CXCL1-CXCR2 axis. TRIM28-recruited MDSCs antagonize effector CD8 + T cells in the tumor immune microenvironment of NSCLC, promoting anti-PD-1 resistance

Article Snippet: Scrambled, human TRIM28 short hairpin RNAs (shRNAs), mouse TRIM28 lentiviral shRNAs viral particles, and mouse TRIM28 lentiviral particles was obtained from Shanghai Genechem Co., Ltd. (Shanghai, China).

Techniques: Injection, Two Tailed Test, Expressing, Activation Assay

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