Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Flow chart of systematic pan-cancer analysis of FBXO5.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques:

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Comparison of FBXO5 expression levels in pan-cancer. (A) FBXO5 expression pattern in 31 forms of normal samples from GTEx database. (B) FBXO5 expression pattern in 30 kinds of tumor cell lines from CCLE database. (C) FBXO5 expression profile in 33 types of cancers from TCGA database.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Comparison, Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Differential analysis of FBXO5 expression in different kinds of tumors. (A) FBXO5 expression differences in tumor tissues from TCGA database compared with normal tissues from GTEx and TCGA databases. (B) Paired differential analysis of FBXO5 expression in matched tumor and normal samples from TCGA. (C) Differential analysis of FBXO5 expression based on cancer patient age in TCGA. (D) Differential analysis of FBXO5 expression based on tumor pathological stages in TCGA. * p -value < 0.05, ** p -value < 0.01, and *** p -value < 0.001. “ns” indicated no significance.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Correlation of FBXO5 expression with survival prognosis in the distinct malignancies studied. (A–D) Forest plots revealing the associations of FBXO5 with OS (A) , DSS (B) , DFI (C) , and PFI (D) in the indicated tumors, respectively. (E–H) Kaplan-Meier curves showing the relationships of FBXO5 expression with OS (E) , DSS (F) , DFI (G) , and PFI (H) in the indicated cancers, respectively.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Association between FBXO5 expression and gene promoter methylation in pan-cancer. (A) Lollipop plot and Pearson’s analyses depicting the correlation between FBXO5 expression and DNA methylation in the indicated tumors. (B–E) Kaplan-Meier curves illustrating the relationships between FBXO5 methylation levels with OS (B) , DSS (C) , DFI (D) , and PFI (E) in the indicated cancers, respectively.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing, Methylation, DNA Methylation Assay

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Association between FBXO5 expression and TME. (A) Heatmap presenting the correlation strength between FBXO5 expression and TME-related terms in pan-cancer. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, and **** p -value < 0.0001. (B–D) Lollipop plots displaying the correlations of FBXO5 expression with ImmuneScore (B) , StromalScore (C) , and ESTIMATEScore (D) in pan-cancer, respectively. (E-G) Pearson’s analyses of the relationships between FBXO5 expression and ImmuneScore (E) , StromalScore (F) , and ESTIMATEScore (G) in the indicated tumor types, respectively.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Correlation between FBXO5 expression and tumor immunity. (A) Heatmap showing the association between FBXO5 expression and the infiltration of immune cells in pan-cancer generated by using the ImmuCellAI database. (B–E) Heatmaps indicating the co-expression of FBXO5 with immune-relevant genes in pan-cancer, including immune activation genes (B) , immunosuppressive genes (C) , chemokine genes (D) , and chemokine-receptor genes (E) . * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, and **** p -value < 0.0001.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing, Generated, Activation Assay

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: Correlations of FBXO5 expression with TMB, MSI, and MMR. (A) Radar map of the association between FBXO5 expression and TMB levels in pan-cancer. The values in blue stand for the range and the lines in red stand for the correlation coefficients. (B) Pearson’s correlation between FBXO5 expression and TMB levels in the indicated cancers. (C) Radar map of the association between FBXO5 expression and MSI frequencies in pan-cancer. (D) Pearson’s correlation between FBXO5 expression and MSI frequencies in the indicated cancers. (E) Heatmap illustrating the association between FBXO5 expression and five MMR genes (MSH2, MSH6, PMS2, MLH1, and EPCAM) in pan-cancer. * p -value < 0.05, ** p -value < 0.01, *** p -value < 0.001, and **** p -value < 0.0001.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: GO annotations of FBXO5 in the indicated six types of tumors using GSEA, including (A) BLCA, (B) BRCA, (C) LIHC, (D) LUAD, (E) OV, and (F) STAD.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques:

Journal: Frontiers in Immunology

Article Title: Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis

doi: 10.3389/fimmu.2022.901784

Figure Lengend Snippet: KEGG annotations of FBXO5 in the indicated six types of tumors using GSEA, including (A) BLCA, (B) BRCA, (C) LIHC, (D) LUAD, (E) OV, and (F) STAD.

Article Snippet: Specifically, the immunohistochemical results based on the antibody against FBXO5 (Cat No. HPA029048; Atlas Antibodies, Sigma-Aldrich) in various normal tissues were downloaded from the tissue section of HPA database, while these data in different tumor tissues were obtained from the pathology section.

Techniques:

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

doi: 10.1038/ncomms6941

Figure Lengend Snippet: (a) ICE2 (NARG2) binds to ELL in the presence of ICE1 (KIAA0947). FLAG-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analyzed by Western blotting. (b) ICE1 binds to EAF1 in the presence of ELL. HA-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analyzed by Western blotting. (c) ICE2 binds to ELL/EAF1 in the presence of ICE1. FLAG-immunopurified complexes from baculovirus-infected Sf9 cells expressing the indicated proteins were analyzed by Western blotting. (d) Proposed architecture of the components of LEC and model for MED26 NTD function as a docking site for LEC. MED26 NTD interacts with LEC through direct interaction with EAF. In turn, the MED26 C-terminal domain binds to Pol II through Mediator. The binding site for MED26 NTD on EAF is represented by black bars. (e) Silver staining of HA-ICE1-CL/FLAG-ICE2/FLAG-ELL/Myc-EAF1 complex reconstituted by the baculovirus expression system. FLAG-ELL, Myc-EAF1, FLAG-ICE2 and HA-ICE1 C-terminal fragment (CL: 1191–2266) were co-expressed in Sf9 insect cells, and the ICE1-CL/ICE2/ELL/EAF1 complex was purified by anti-HA affinity chromatography as described in the Methods section. * indicates heavy chain and light chain derived from anti-HA antibodies used in affinity purification. (f) ICE1-CL/ICE2/ELL/EAF1 complex enhances transcription elongation by Pol II. Oligo(dC)-tailed template transcription assays were performed as described in the Results and Methods sections. Arrowhead indicates the position of the nascent transcript that is synthesized in the presence of ATP, GTP and CTP and stalled at the T site.

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz), MED23 (A300-425A-1, Bethyl Laboratories, Montgomery, TX), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1865–2266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa cruz), Rpb1 (N20, sc-899, Santa Cruz) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, California).

Techniques: Infection, Expressing, Western Blot, Binding Assay, Silver Staining, Purification, Affinity Chromatography, Derivative Assay, Affinity Purification, Synthesized

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

doi: 10.1038/ncomms6941

Figure Lengend Snippet: (a, b, c and d) Depletion of MED26 decreases the occupancy of ICE1 (KIAA0947) at a subset of Pol II-transcribed snRNA genes. ChIP-sequence analysis was performed using triplicate ChIP samples. Genome browser track examples showing the effect of MED26 depletion on ICE1 occupancy at a subset of snRNA genes including RNU4-1 and RNU4-2 (a), RNU5A and RNU5B (b), RNU11 (c) and RNU12 (d) were depicted by averaging three of each sequence read from triplicate ChIPs. (e) Western blot showing Mediator, Pol II and LEC components in HEK293T cells treated with non-targeting siRNA or MED26 siRNA#1. (f) Knockdown of MED26 decreases the occupancy of ICE1, ELL, EAF2, and total and Ser7-phosphorylated Pol II at a subset of snRNA genes. Ct values of each ChIP were normalized to that of input. Each value is the average of three independent experiments, and error bars show standard deviation (s.d.). The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.05; **, P < 0.01).

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz), MED23 (A300-425A-1, Bethyl Laboratories, Montgomery, TX), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1865–2266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa cruz), Rpb1 (N20, sc-899, Santa Cruz) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, California).

Techniques: Sequencing, Western Blot, Knockdown, Standard Deviation

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

doi: 10.1038/ncomms6941

Figure Lengend Snippet: (a) MED26 depletion in cells decreases the expression of a subset of Pol II-transcribed snRNA and snoRNA genes. HEK293T cells were transfected with non-targeting siRNA as a control and each of three different siRNAs (#1, #2 and #3) targeting MED26. Total RNAs were extracted from cells and the expression of indicated genes was measured by real-time qPCR. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.04; **, P < 0.008). (b) MED26 wild type (wt) contributes more to the expression of a subset of snRNA genes than does a MED26 point mutant (mut: R61A, K62A). Total RNAs were extracted from MED26 gene-trapped MEF cells expressing HA-tagged MED26 wild type (wt) or point mutant (mut), and the expression of the indicated genes was measured by real-time qPCR. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.04; **, P < 0.008).

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz), MED23 (A300-425A-1, Bethyl Laboratories, Montgomery, TX), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1865–2266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa cruz), Rpb1 (N20, sc-899, Santa Cruz) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, California).

Techniques: Expressing, Transfection, Control, Mutagenesis

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

doi: 10.1038/ncomms6941

Figure Lengend Snippet: (a) Western blot showing TAF7 and Hsp90 at 48 h after transient transfection of non-targeting (control) siRNA pool or siRNA pool targeting TAF7. (b) Effect of TAF7 depletion on transcript levels of Pol II-transcribed snRNA genes detected by RT-qPCR performed with total RNA from cells transfected with siRNAs. Ct values were normalized to GAPDH. Data points are the average of three independent experiments, and error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.05; **, P < 0.01). (c) Schematic representation of immobilized oligo(dC)-tailed template assay. Templates immobilized on Streptavidin beads were incubated with purified recombinant protein EAF1 in the presence or absence of Mediator and/or TAF7 N243. After washing, bead-bound proteins were detected by Western blotting. (d) TAF7 interferes with EAF1 recruitment by Mediator in vitro. Upper panel shows immobilized oligo(dC)-tailed template assays. Assays were performed with an oligo(dC)-tailed template, EAF1, and Mediator in the presence or absence of TAF7 N243. Lower panel shows silver staining of Mediator and HA-tagged TAF7 N243 used in the assay. Mediator (x1) and HA-TAF7 N243 (x1) contain 1 pmol of the each protein or protein complex. (e) Model of immobilized oligo(dC)-tailed template assay. TAF7 N243 inhibits EAF1 recruitment to the oligo(dC)-tailed template by MED26-containing Mediator. (f) Depletion of TAF7 increased the occupancy of ICE1 (KIAA0947), EAF2, and total Pol II at U4-1 and U5B snRNA genes. Ct values of each ChIP were normalized to that of input. Data points are the average of three independent experiments; error bars show s.d.. The P values for the indicated comparisons were determined by Student’s t test (*, P < 0.05; **, P < 0.01).

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz), MED23 (A300-425A-1, Bethyl Laboratories, Montgomery, TX), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1865–2266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa cruz), Rpb1 (N20, sc-899, Santa Cruz) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, California).

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Incubation, Purification, Recombinant, In Vitro, Silver Staining

Journal: Nature communications

Article Title: MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex

doi: 10.1038/ncomms6941

Figure Lengend Snippet: In the initiation or early elongation stage, TAF7 interacts with MED26 NTD and blocks LEC recruitment by MED26. In the transition from initiation to elongation, MED26 NTD is released from TAF7 and LEC is recruited to a subset of snRNA genes. After LEC is recruited by MED26, MED26 hands LEC to Pol II, and thereby LEC activates productive elongation of Pol II. Binding sites for the MED26 NTD on TAF7 and EAF family members are represented by black bars. N, MED26 NTD.

Article Snippet: Specific antibodies used were as follows: MED26 (H-228, sc-48776 X, Santa Cruz), MED23 (A300-425A-1, Bethyl Laboratories, Montgomery, TX), ICE1 (raised against bacterially expressed, His-tagged recombinant proteins of ICE1 1865–2266 aa), ELL (A301-645A, Bethyl Laboratories), EAF2 (A302-502A-1, Bethyl Laboratories), TAF7 (SQ-8, sc-101167, Santa cruz), Rpb1 (N20, sc-899, Santa Cruz) and RNA Pol II CTD phospho Ser7 (Cat# 61087, Active motif, Carlsbad, California).

Techniques: Binding Assay