Journal: Nature metabolism

Article Title: Genetic susceptibility to diabetic kidney disease is linked to promoter variants of XOR

doi: 10.1038/s42255-023-00776-0

Figure Lengend Snippet: a, Promoter luciferase assay performed on 293T cells with luciferase reporter constructs containing the Xor variants sequence upstream of the transcription start site; D-13, in normal glucose (NG; 5 mM) with control vector, mannitol (25 mM) or high glucose (HG; 30 mM) with D-13 vector (n = 3 biologically independent experiments; data are presented as mean values ± s.e.m.). b, Top, ChIP was carried out in kidney cortex tissue from diabetic B6 and D2 mice (n = 3 mice/group) using normal rabbit IgG antibodies, anti-STAT6 antibodies or anti-C/EBPβ antibodies, as indicated. Precipitated DNA was analysed by amplifying the PCR product in the Xor promoter, which is approximately 300 bp long. IgG served as a negative control. Bottom, a schematic of the C/EBPβ binding site in the sequence of the Xor promoter. c,d, Immunohistochemistry staining of C/EBPβ in diabetic B6 (c) and diabetic D2 (d) kidneys, showing nuclear staining in glomeruli (filled arrows) and some peritubular capillaries (open arrows). ×40 magnification; scale bars, 100 μm. e, Heatmap showing the genes that are differentially regulated by C/EBPβ in non-diabetic (n = 5) and diabetic D2 mice (n = 3); the arrow indicates Xdh as a top gene under the regulation of C/EBPβ in diabetes (P < 9.41 × 10−17). f, Cebpb mRNA in mGECs treated with a scrambled siRNA (Scrbl) or siRNA targeting Cebpb for 24 h (n = 7 biologically independent experiments, data are presented as mean values ± s.e.m.). g, Xor mRNA (bar graph) and protein (blot) expression in mGECs under the control condition NG or the HG condition, treated with scrambled or Cebpb-targeting siRNA for 24 h. h, Percentage of mGECs expressing CM-H2-DCFDA, a marker for ROS. mGECs were kept in NG or HG and were treated with scrambled or Cebpb siRNA for 24 h (n = 5 and n = 4 independent experiments in g and h, respectively; data are presented as mean values ± s.e.m.). To determine statistical significance, unpaired two-tailed Student’s t-tests were performed in f, and a one-way ANOVA with Tukey’s multiple-comparisons test was performed in a, g and h.

Article Snippet: Gene silencing RNA interference experiments in cells were performed after transfection of siRNA targeting Cebpb (Oligo Duplex (Locus ID 12608), OriGene) or with a scramble sequence, using Lipofectamine RNAiMAX (Invitrogen).

Techniques: Luciferase, Construct, Sequencing, Control, Plasmid Preparation, Negative Control, Binding Assay, Immunohistochemistry, Staining, Expressing, Marker, Two Tailed Test

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: WT p53 suppresses mRNA and protein levels of C/EBPβ in CRC cell lines. (A) Protein and mRNA expression of C/EBPβ in CT26 cells stably expressing shCtrl or sh Trp53 were analyzed by Western blotting and real-time PCR, respectively. (B) C/EBPβ protein expression was determined in CRC cell lines harboring TP53 WT, nonsense mutation, and missense mutation treated with nutlin-3a (10 μmol/l) for WT p53 induction or vehicle for 24 h (left), as well as CEBPB mRNA levels in TP53 WT cell lines (right). (C) C/EBPβ protein expression was compared in TP53 WT HCT116 and SNU1544 cells after transfection with siCtrl or si TP53 , followed by nutlin-3a (10 μmol/l) or vehicle treatment for 24 h. (D) C/EBPβ protein expression was determined in TP53 nonsense SNUC1 and SNU1411 cells after transient transfection with EV or TP53 WT, followed by 24 h of nutlin-3a (10 μmol/l) treatment. (E) C/EBPβ protein levels were determined in SNU1544 cells pretreated with nutlin-3a (10 μmol/l) for 16 h and stimulated with 8-Br-cAMP (100 μmol/l), an inducer of CEBPB transcription, for 8 h. (F) C/EBPβ protein levels were determined in SNU1411 cells transiently transfected with EV, TP53 WT, or TP53 R175H for 24 h. (G) C/EBPβ protein (left) and mRNA (right) levels were assessed in p53-null Caco-2 cells stably transduced with doxycycline-inducible TP53 WT or TP53 R175H following treatment with 1 μg/ml doxycycline for 48 h. (H) C/EBPβ protein stability was compared in shCtrl and sh Trp53 CT26 cells after CHX treatment (5 μg/ml) for the indicated times. (I) SNU1544 were treated with nutlin-3a (10 μmol/l) for 21 h, followed by MG132 (2 μmol/l) for 3 h to block proteasomal degradation. Protein expression was analyzed by Western blotting, representative of 3 independent experiments, and the values presented underneath or in the graph (H) were generated by densitometric analyses using ImageJ. mRNA levels were determined using real-time PCR. The bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: 8-Br-cAMP, 8-bromoadenosine 3′,5′-cyclic monophosphate; CEBPB , CCAAT enhancer binding protein beta; CHX, cycloheximide; CRC, colorectal cancer; doxy, doxycycline; EV, empty vector; SD, standard deviation; shCtrl, control short hairpin RNA; sh Trp53 , short hairpin RNA for Trp53 ; si TP53 , small interfering RNA for TP53 ; WT, wild type.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: Expressing, Stable Transfection, Western Blot, Real-time Polymerase Chain Reaction, Mutagenesis, Transfection, Transduction, Blocking Assay, Generated, Binding Assay, Plasmid Preparation, Standard Deviation, Control, shRNA, Small Interfering RNA

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: Tumor cell C/EBPβ increases CTLA-4 expression in CD4 + T cells. (A) CT26 cells expressing Cebpb -oe and EV control were injected subcutaneously into immunocompetent BALB/c mice ( n = 4; representative of 3 independent experiments, top) and immunodeficient BALB/c nude mice ( n = 5; single experiment, bottom). (B) Subcutaneous tumors from EV and Cebpb -oe CT26 cells injected into BALB/c mice were collected approximately 21 days post-inoculation. The percentages of CD4 + and CD8 + cells among CD45 + cells, CTLA-4 + cells among CD4 + and CD8 + cells, Foxp3 + CTLA-4 + and Foxp3 − CTLA-4 + cells among CD4 + cells, and GzmB + and IFN-γ + cells among CD8 + cells were compared by flow cytometry. (C) Mouse splenic T cells isolated from BALB/c mice were activated with anti-CD3ε and anti-CD28, then co-cultured with EV or Cebpb -oe CT26 cells for 72 h. The culture ratio of T cells to CT26 cells is indicated. Flow cytometry was used to analyze CTLA-4 + and Foxp3 + levels among CD4 + cells, as well as CTLA-4 + , PD-1 + , GzmB + , and IFN-γ levels among CD8 + cells. Representative histograms of CTLA-4 + expression in CD4 + T are presented. (D) mRNA expression of immune checkpoint genes in Jurkat (left) and EL4 cells (right) after co-culture with indicated cells, measured by real-time PCR. (E) Proliferation analysis of CFSE-labeled, activated mouse splenic T cells co-cultured with indicated cells for 72 h. CFSE dilution in CD4 + T and CD8 + T cells were assessed by flow cytometry and presented as histograms (representative of 2 independent experiments). (F) Transwell migration assay of mouse splenic T cells (left) and EL4 (right) cultured for 24 h in CM from Cebpb -oe or EV CT26 cells or in fresh media, in the presence of CXCL12 (200 ng/ml). (G) Tumor growth curves of BALB/c mice bearing Cebpb -oe CT26 tumors treated with isotype control IgG, anti-PD-1, anti-CTLA-4, or the combination ( n = 5 per group). Tumor volumes are shown as mean ± SD; statistical comparisons were made using 2-tailed Student’s t tests based on the final tumor volume. Bar graphs represent the mean ± SD; each dot represents an independent biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: CEBPB , CCAAT enhancer binding protein beta; Cebpb -oe, Cebpb -overexpression; CFSE, carboxyfluorescein succinimidyl ester; CM, conditioned media; CTLA-4, cytotoxic T-lymphocyte associated protein 4; CXCL12, C-X-C motif chemokine ligand 12; EV, empty vector; Foxp3, forkhead box protein P3; LAG3 , lymphocyte activating 3; PD-1, programmed cell death protein 1; PDCD1 , programmed cell death 1; SD, standard deviation.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: Expressing, Control, Injection, Flow Cytometry, Isolation, Cell Culture, Co-Culture Assay, Real-time Polymerase Chain Reaction, Labeling, Transwell Migration Assay, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation

Journal: Cancer Communications

Article Title: CEBPB Expression in Tumor Cells Drives Immune Evasion in Colorectal Cancer via CTLA4 Up-regulation in T Cells

doi: 10.34133/cancomm.0013

Figure Lengend Snippet: Lipocalin-2 mediates C/EBPβ-driven CTLA-4 up-regulation in T cells. (A) Venn diagram of genes up-regulated in CEBPB -oe compared to EV cells in CT26, SNU1544, and SNU81, analyzed by RNA sequencing, identifying 13 genes that were concurrently up-regulated. (B) CM from EV or Cebpb -oe CT26 cells were subjected to mouse cytokine array. Each cytokine is represented by duplicate spots, and those showing altered expression are indicated (left). The graph shows signal intensity relative to control spots, analyzed by densitometric quantification (right). (C) Protein levels of C/EBPβ and lipocalin-2 in SNU1544 and SW837 cells by Western blotting . (D) mRNA expression of CTLA4 in Jurkat cells after 24 h co-culture with indicated cells, measured by real-time PCR. (E) mRNA expression of immune checkpoint genes in Jurkat cells after co-culture with LCN2 -oe or EV SNU1544 cells. (F) mRNA expression of CTLA4 in Jurkat cells co-cultured with SW837 cells at a 1:1 ratio in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h. (G) Protein levels of CEBP/β and lipocalin-2 in CEBPB -oe and EV SNU1544 cells (left). mRNA levels of CTLA4 in Jurkat cells co-cultured with CEBPB -oe or EV SNU1544 cells in the presence of lipocalin-2 neutralizing antibody (1 μg/ml) or isotype IgG control for 24 h (right). (H) Protein levels of C/EBPβ and lipocalin-2 in EV, Cebpb -oe, and Lcn2 -oe CT26 cells (left). Flow cytometry analysis of CTLA-4 + in CD4 + and CD8 + cells from activated mouse splenic T cells co-cultured with EV or Lcn 2-oe CT26 cells for 72 h at the indicated culture ratio of T cells to CT26 cells (right). (I) Flow cytometry analysis of activated mouse splenic T cells co-cultured with Cebpb -oe CT26 at a 5:1 ratio for 48 h in the presence of lipocalin-2 neutralizing antibody (2 μg/ml) or isotype IgG control. The bar graphs represent the mean ± SD; each dot indicates a biological replicate. P values were calculated using 2-tailed Student’s t tests. Abbreviations: AKR1C1 , aldo-keto reductase family 1 member C1; C3 , complement C3; CDA , cytidine deaminase; CD14 , cluster of differentiation 14; CEBPB , CCAAT enhancer binding protein beta; CEBPB -oe, CEBPB overexpression; CM, conditioned media; CTLA4 , cytotoxic T-lymphocyte associated protein 4; CXCL1, C-X-C motif chemokine ligand 1; EV, empty vector; IL1RL1 , interleukin 1 receptor like 1; IRF7 , interferon regulatory factor 7; LCN2 , lipocalin-2; Lcn2 -oe, Lcn2 -overexpression; S100A8 , S100 calcium binding protein A8; S100A9 , S100 calcium binding protein A9; SBSN , suprabasin; SD, standard deviation; SERPINB2 , serpin family B member 2; SLPI , secretory leukocyte peptidase inhibitor.

Article Snippet: For overexpression of genes, cDNA of human TP53 and LCN2 from HEK293T and mouse Lcn2 from CT26 were generated by PCR amplification, cDNA was obtained from human CEBPB (SC319561, OriGene) and mouse Cebpb (MR227563, OriGene), open reading frame clones were cloned into entry vector pDONR221 (12536017, Thermo Fisher Scientific), and entry vector with non-transcribed stuffer sequence (C125-E01, a gift from Dominic Esposito, Frederick National Laboratory for Cancer Research) was used for empty vector (EV) controls.

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Binding Assay, Over Expression, Plasmid Preparation, Standard Deviation