murine cebpb  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A) Venn diagram of differentially expressed transcription factors from GEO/TCGA databases. (B-D) Heatmaps of 5 consistently dysregulated transcription factors (red: upregulated; green: downregulated). (E-G) CEBPB mRNA expression in different patient cohorts: UC patients (GSE87466: 21 healthy individuals vs 87 UC; Welch’s two-tailed t-test); CAC patients (GSE37283: 5 healthy individuals vs 11 CAC; unpaired t-test); and CRC patients (TCGA: 51 normal adjacent tumor (NAT) vs 383 tumors; Mann-Whitney U test). (H) Kaplan-Meier survival analysis of 376 CRC patients stratified by CEBPB mRNA expression (TCGA). Data are expressed as mean ± SD. *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A) Representative hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining of C/EBPβ in normal colon, CAC-adjacent (CAC-AT), and CAC tissues (100×: 100 μm scale; 400×: 25 μm scale). (B-C) Quantitative analysis of (B) C/EBPβ protein (IHC) and (C) C EBPB mRNA levels (normal: n = 10; CAC: n = 5; one-way ANOVA with Bonferroni correction). (D) CRC tissue microarrays (n = 180) showing C/EBPβ expression (40×: 250 μm scale; 200×: 50 μm scale). (E) C/EBPβ IHC scores across CRC Stages (Kruskal-Wallis with Dunn’s test). (F) Kaplan-Meier survival analysis of 180 CRC patients by C/EBPβ expression. (G-H) Western blot analyses of C/EBPβ expression in randomly selected CRC patients (n = 12). Protein levels were quantified by densitometry (normalized to GAPDH) using ImageJ software. Statistical analysis was conducted using the Mann-Whitney U test. (I) qRT-PCR was employed to evaluate CEBPB mRNA levels in randomly selected CRC patients (n = 64). Statistical analysis was performed using the Mann-Whitney U test. Data are expressed as mean ± SD. * P < 0.05, *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Immunohistochemistry, Expressing, Western Blot, Software, MANN-WHITNEY, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A) Representative images of colons from untreated wild-type (WT) control mice and AOM/DSS-treated WT mice. (B) Cebpb mRNA levels in colonic tissue from WT control and AOM/DSS-treated WT mice were quantified by qRT-PCR. Statistical analysis was performed by Mann-Whitney U test; n = 5 mice/group. (C) Western blot analysis of C/EBPβ protein expression in colonic epithelium and stroma of WT control and AOM/DSS-treated WT mice. Protein levels were normalized to GAPDH. (D) Representative images of colons from three experimental groups: (i) WT control mice, (ii) AOM/DSS-treated WT mice (WT AOM/DSS), and (iii) AOM/DSS-treated mice with intestinal epithelial cell-specific Cebpb knockout ( Cebpb ΔIEC AOM/DSS). Tumor number and size were assessed; n = 5 mice/group. (E) Colon tumors were quantified by size stratification (>2 mm, measured by calipers; <2 mm, assessed by dissection microscope). Data were analyzed by one-way ANOVA with Bonferroni’s multiple comparisons test; n = 5 mice/group. (F) Representative H&E and IHC staining of C/EBPβ in colon tissues from WT control, WT AOM/DSS and Cebpb ΔIEC AOM/DSS mice. Scale bar = 100 μm. (G) Quantification of C/EBPβ-positive staining areas in colon tissues from WT control, WT AOM/DSS and Cebpb ΔIEC AOM/DSS mice by ImageJ software. (H-I) C/EBP β mRNA levels (qRT-PCR) and protein expression (Western blot) in colon tissues from WT control, WT AOM/DSS and Cebpb ΔIEC AOM/DSS mice. Statistical analysis was performed by Kruskal-Wallis test with Dunn’s multiple comparisons test. n = 5 mice/group for qRT-PCR. Data are presented as mean ± SD. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Control, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Knock-Out, Dissection, Microscopy, Immunohistochemistry, Staining, Software

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A-C) Immune cell subsets in colon tissues from WT control, WT AOM/DSS, and Cebpb ΔIEC AOM/DSS mice were quantified by flow cytometry (n = 3 mice/group). Data analyzed by one-way ANOVA with Bonferroni’s correction. (D-I) Bioinformatics analysis of neutrophil-associated genes in human datasets (Mann-Whitney U test): For panels D-E, data were obtained from 26 healthy individuals and 11 CAC patients (GSE87466, GSE37283). For panels F-G, data were obtained from 41 normal adjacent tumor tissues (NAT) and 216 CRC samples (TCGA-COAD/READ). For panels H-I, data were collected from 21 healthy individuals and 87 UC patients (GSE87466 dataset). Data are expressed as mean ± SD. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Control, Flow Cytometry, MANN-WHITNEY

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A-D) KEGG and gene set enrichment analysis (GSEA) of differentially expressed genes (DEGs) in colon tissues from WT control, WT AOM/DSS, and Cebpb ΔIEC AOM/DSS mice (n = 5 mice /group). (E-F) Heatmap showing 36 significantly enriched DEGs from the cytokine-cytokine receptor interaction pathway (Z-scores shown by color gradient). (G-H) CXCR2 ligand (CXCL1/2/5) and receptor levels in colon tissues measured by qRT-PCR (G; n = 5) and ELISA (H; n = 5. Statistical analysis was performed by one-way ANOVA with Bonferroni’s correction. (I-J) CXCL1/2/5 expression in Caco-2 cells with CEBPB overexpression (pcDNA3.1- CEBPB ) or knockdown ( CEBPB -siRNA), compared to their respective controls (one-way ANOVA with Bonferroni’s correction). (K) ChIP-qPCR confirming C/EBPβ binding to CXCL1/2/5 promoters (anti-C/EBPβ antibody vs. Input/IgG controls; unpaired t-test). (L) Luciferase reporter assays of CEBPB- responsive promoter activity (wild-type vs. mutant CXCL1/2/5 promoters) in CEBPB -overexpressing Caco-2 cells (unpaired t-test). (M-N) Representative images showing neutrophil migration toward supernatants from CEBPB -modulated Caco-2 cells (SB225002-pretreated; 100× magnification, scale bar = 100 μm; one-way ANOVA with Bonferroni’s correction) n = 3 biological replicates for panels I-N. Data are expressed as mean ± SD. ns: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Over Expression, Knockdown, ChIP-qPCR, Binding Assay, Luciferase, Activity Assay, Mutagenesis, Migration

Journal: bioRxiv

Article Title: Intestinal Epithelial C/EBPβ Deficiency Impairs Colitis-Associated Tumorigenesis by Disrupting CXCL1/CXCL2/CXCL5-CXCR2-Mediated Neutrophil Infiltration

doi: 10.1101/2025.06.25.661448

Figure Lengend Snippet: (A) Colons specimens were harvested from four groups: (i) WT AOM/DSS, (ii) Cebpb ΔIEC AOM/DSS, and (iii-iv) SB225002 (CXCR2 antagonist)-treated counterparts (WT or Cebpb ΔIEC AOM/DSS mice). (B) Tumor burden quantification (>2 mm by calipers; <2 mm by dissection microscopy; one-way ANOVA with Bonferroni’s correction). n = 5 mice/group. (C) Representative H&E staining in colon tissues from four groups (100×, 100 μm scale). (D-E) Flow cytometry analysis of CD11b + Ly-6G + neutrophils among CD45 + leukocytes (one-way ANOVA with Bonferroni’s correction). (F-G) Immunofluorescence visualization (200×, 50 μm scale) and ImageJ quantification of Ly-6G + CD11b + cell infiltrate in 5 high-power fields (HPF). n = 5 mice/group. Data are expressed as mean ± SD. ns: not significant, *** P < 0.001.

Article Snippet: Reagents included AOM and SB225002 (Sigma, St. Louis, USA), DSS (MP Biomedicals, Solon, USA), CEBPB siRNA from Thermo Fisher (Grand Island, USA) (Supplementary Table S7), and a pcDNA3.1- CEBPB plasmid (Real Gene, Nanjing, China).

Techniques: Dissection, Microscopy, Staining, Flow Cytometry, Immunofluorescence

Journal: Open Life Sciences

Article Title: CEBPB promotes ulcerative colitis-associated colorectal cancer by stimulating tumor growth and activating the NF-κB/STAT3 signaling pathway

doi: 10.1515/biol-2022-1012

Figure Lengend Snippet: CEBPB affects the progression of UCCRC mice: (a) the modeling method of AOM/DSS-induced UCCRC in mice and treatment regimens, (b) representative photographs of AOM/DSS-induced UCCRC in mice, (c) colorectal length of mice, (d) colon weight of mice, (e) survival rate of mice, and (f) body weight of mice. N = 10, * P < 0.05.

Article Snippet: Recombinant adeno-associated virus serotype 9 (rAAV9) expressing a siRNA targeting CEBPB (siCEBPB), full-length CEBPB cDNA (CEBPB), and scramble control (NC) were generated by Biowit Technologies Co., Ltd. (Shenzhen, China), according to the manufacturer’s protocol.

Techniques:

Journal: Open Life Sciences

Article Title: CEBPB promotes ulcerative colitis-associated colorectal cancer by stimulating tumor growth and activating the NF-κB/STAT3 signaling pathway

doi: 10.1515/biol-2022-1012

Figure Lengend Snippet: CEBPB affects the pathogenic alterations and inflammatory response in UCCRC mice, and representative images of H&E-stained colorectal tissues of mice. The scale bar is 200 μm (a); the concentrations of TNF-α (b), IL-6 (c), and IL-1β (d) in mice’s sera were determined with ELISA kits; the expression of TNF-α (e), IL-6 (f), and IL-1β (g) in colon tissues was determined with RT-qPCR assay, as per the manufacturer’s protocol. * P < 0.05. The red arrows represent the pathological changes.

Article Snippet: Recombinant adeno-associated virus serotype 9 (rAAV9) expressing a siRNA targeting CEBPB (siCEBPB), full-length CEBPB cDNA (CEBPB), and scramble control (NC) were generated by Biowit Technologies Co., Ltd. (Shenzhen, China), according to the manufacturer’s protocol.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Open Life Sciences

Article Title: CEBPB promotes ulcerative colitis-associated colorectal cancer by stimulating tumor growth and activating the NF-κB/STAT3 signaling pathway

doi: 10.1515/biol-2022-1012

Figure Lengend Snippet: CEBPB alters the expression of the related proteins and the activation of NF-κB/STAT3 signaling pathway, IHC analysis of the protein expression of E-cadherin (a) and Ki67 (b) in the UCCRC tissues of mice. The scale bar is 50 μm and 100 μm. (c)–(g) Western blot analysis of the protein expression of Ki67, E-cadherin, N-cadherin, and vimtein in the UCCRC tissues of mice. RT-qPCR analysis of the mRNA expression of Ki67 (h), E-cadherin (i), N-cadherin (j), and vimentin (k) in UCCRC tissues of mice. (l) Western blot analysis of p-STAT3 and NF-κB-p65 expression UCCRC tissues of mice. (m) Western blot analysis of p-STAT3 expression in SW480 cells treatment with PMA. After PMA or Colivelin TFA treats SW480 cells, clone formation, transwell, and scratch-wound assay were used to detect cell proliferation (n), invasion (o), and migration (p). N = 3, * P < 0.05. PMA, phorbol 12-myristate 13-acetate; the red arrows represent the pathological changes.

Article Snippet: Recombinant adeno-associated virus serotype 9 (rAAV9) expressing a siRNA targeting CEBPB (siCEBPB), full-length CEBPB cDNA (CEBPB), and scramble control (NC) were generated by Biowit Technologies Co., Ltd. (Shenzhen, China), according to the manufacturer’s protocol.

Techniques: Expressing, Activation Assay, Western Blot, Quantitative RT-PCR, Scratch Wound Assay Assay, Migration

Journal: Open Life Sciences

Article Title: CEBPB promotes ulcerative colitis-associated colorectal cancer by stimulating tumor growth and activating the NF-κB/STAT3 signaling pathway

doi: 10.1515/biol-2022-1012

Figure Lengend Snippet: CEBPB activates the NF-κB/STAT3 signaling pathway in UCCRC mice: (a) Western blot analysis of the protein expression of NF-κB-p65 and p-STAT3 in the UCCRC tissues of mice. (b) Representative photographs of the different groups in mice and (c) the colon weight of mice, (d) colorectal length of mice, and (e) the survival rate of mice. (f) Representative images of H&E-stained colorectal tissues of mice. The scale bar is 200 μm. (g) The concentrations of TNF-α, IL-6, and IL-1β in mice’s sera were determined with ELISA kits. (h) The expression of TNF-α, IL-6, and IL-1β in colon tissues was determined with RT-qPCR assay, as per the manufacturer’s protocol. * P < 0.05. The red arrows represent the pathological changes.

Article Snippet: Recombinant adeno-associated virus serotype 9 (rAAV9) expressing a siRNA targeting CEBPB (siCEBPB), full-length CEBPB cDNA (CEBPB), and scramble control (NC) were generated by Biowit Technologies Co., Ltd. (Shenzhen, China), according to the manufacturer’s protocol.

Techniques: Western Blot, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Open Life Sciences

Article Title: CEBPB promotes ulcerative colitis-associated colorectal cancer by stimulating tumor growth and activating the NF-κB/STAT3 signaling pathway

doi: 10.1515/biol-2022-1012

Figure Lengend Snippet: CEBPB regulates the expression of proteins via activating NF-κB/STAT3 signaling pathway, IHC analysis of the protein expression of E-cadherin and Ki67 in the UCCRC tissues of mice. The scale bar is 50 μm. (b) Western blot analysis of the protein expression of Ki67, E-cadherin, N-cadherin, and vimentin in the UCCRC tissues of mice. (c) Real-time RT-qPCR analysis of the mRNA expression of Ki67, E-cadherin, N-cadherin, and vimentin in colon tissues of mice. N = 3, * P < 0.05. The red arrows represent the pathological changes.

Article Snippet: Recombinant adeno-associated virus serotype 9 (rAAV9) expressing a siRNA targeting CEBPB (siCEBPB), full-length CEBPB cDNA (CEBPB), and scramble control (NC) were generated by Biowit Technologies Co., Ltd. (Shenzhen, China), according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: Expression of LRRC8A in M 2 -MACs. ( A ) Real-time PCR examination of LRRC8A and ANO1 in M 2 -MACs. Expression levels are shown as the ratio to the ACTB ( n = 4 for each). ( B ) Protein expression of LRRC8A (a) and ANO1 (b) in M 2 -MACs protein lysates. Blots were probed with anti-LRRC8A (approx. 95 kDa), anti-ANO1 (approx. 115 kDa), and anti-ACTB (approx. 45 kDa) antibodies. ( C ) Confocal fluorescence images (green) of Alexa Fluor 488-labeled LRRC8A (b) and ANO1 (c) and negative control with Alexa Fluor 488 alone (a) in M 2 -MACs. Dashed lines show cell boundaries. The scale bar in ‘(a)’ shows 10 μm (same in (b,c)).

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Labeling, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: Functional expression of LRRC8A in M 2 -MACs. ( A , B ) Simultaneous measurement of changes in membrane potential ( A ) and [Ca 2+ ] i ( B ) following the application of the LRRC8/ANO1 inhibitor, endovion (EDV, 1 μM), using bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC 4 (3)] and Fura 2-acetoxymethyl ester (Fura 2-AM), respectively. The relative time course of changes in fluorescence intensities (1.0 at time 0 s) from an M 2 -MAC is shown. ( C , D ) Summarized results of EDV (1 μM)-induced depolarization (at 1 min) ( C ) and hyperpolarization (at 10 min) ( D ) responses. ( E , F ) Summarized results of EDV (1 μM)-induced changes in [Ca 2+ ] i at 1 min ( E ) and 10 min ( F ). ( G , H ) Measurement of changes in membrane potential following the application of the selective ANO1 inhibitor, ANO1-IN-1 (1 μM), using DiBAC 4 (3). The relative time course of changes in fluorescence intensities (1.0 at time 0 s) from an M 2 -MAC is shown ( G ). Summarized results of ANO1-IN-1-induced responses ( H ). Numbers used for experiments are shown in parentheses. **: p < 0.01 vs. the vehicle control.

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Functional Assay, Expressing, Membrane, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: Effects of the siRNA-mediated and pharmacological inhibition of LRRC8A on IL-8 and IL-10 expression and secretion in M 2 -MACs. ( A , C ) Real-time PCR examination of IL-8 ( A ) and IL-10 ( C ) expression in M 2 -MACs 48 h after the transfection of LRRC8A siRNA (siLRRC8A). The mRNA expression level in the control siRNA (siCont)-transfected group is expressed as 1.0 ( n = 4 for each). ( B , D ) Quantitative detection of IL-8 ( B ) and IL-10 ( D ) secretion by an ELISA assay in the siCont and siLRRC8A groups. The cytokine secretion level in the siCont group is expressed as 1.0 ( n = 4 for each). ( E , G ) Real-time PCR examination of IL-8 ( E ) and IL-10 ( G ) expression in vehicle- and endovion (EDV: 1, 3, and 10 μM)-treated M 2 -MACs for 12 h. The mRNA expression level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( F , H ) Quantitative detection of IL-8 ( F ) and IL-10 ( H ) secretion by an ELISA assay in the vehicle- and EDV (10 μM)-treated groups. The cytokine secretion level in the vehicle control is expressed as 1.0 ( n = 4 for each). *, **: p < 0.05, 0.01 vs. siCont and the vehicle control.

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: No involvement of AKT or AMPK in the LRRC8A inhibition-induced downregulation of IL-8 and IL-10 in M 2 -MACs. ( A ) Protein expression of P-Nrf2, Nrf2, P-AKT, and AKT in protein lysates of M 2 -MACs. Blots were probed with anti-P-Nrf2/Nrf2 (approx. 100 kDa), anti-P-AKT/AKT (approx. 60 kDa), and anti-ACTB (approx. 45 kDa) antibodies. ( B , C ) Real-time PCR examination of IL-8 ( B ) and IL-10 ( C ) expression in vehicle- and AKT inhibitor AZD5363 (2 μM)-treated M 2 -MACs for 12 h. The mRNA level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( D ) Protein expression of P-AMPK and AMPK in protein lysates of vehicle- and endovion (EDV; 10 μM)-treated M 2 -MACs for 2 h. Blots were probed with anti-P-AMPK/AMPK (approx. 65 kDa) and anti-ACTB (approx. 45 kDa) antibodies. ( E ) Summarized results of the relative expression of P-AMPK/AMPK were obtained from the optical density of P-AMPK, AMPK, and ACTB band signals ( n = 4 for each).

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: Involvement of NOX2 in the LRRC8A inhibition-induced downregulation of IL-8 and IL-10 in M 2 -MACs. ( A ) Identification of NOX isoforms mainly expressed in M 2 -MACs by a real-time PCR examination. Expression levels are shown as the ratio to the ACTB. The mRNA expression level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( B ) Protein expression of NOX2 in M 2 -MAC protein lysates. Blots were probed with anti-NOX2 (approx. 60 kDa) and anti-ACTB (approx. 45 kDa) antibodies. ( C – E ) Real-time PCR examination of IL-8 ( C ), IL-10 ( D ), and CEBPB ( E ) in vehicle- and NOX4/NOX2 inhibitor GLX351322 (GLX) (10 and 100 μM)-treated M 2 -MACs for 12 h. The mRNA expression level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( F , G ) Quantitative detection of IL-8 ( F ) and IL-10 ( G ) secretion by an ELISA assay in vehicle- and GLX (100 μM)-treated M 2 -MACs for 24 h. The cytokine secretion level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( H – J ) Real-time PCR examination of IL-8 ( H ), IL-10 ( I ), and CEBPB ( J ) in vehicle- and selective NOX2 inhibitor GSK2796039 (GSK) (10 μM)-treated M 2 -MACs for 12 h. The mRNA expression level in the vehicle control is expressed as 1.0 ( n = 4 for each). ( K , L ) Quantitative detection of IL-8 ( K ) and IL-10 ( L ) secretion by an ELISA assay in vehicle- and GSK-treated M 2 -MACs for 24 h. The cytokine secretion level in the vehicle control is expressed as 1.0 ( n = 4 for each). **: p < 0.01 vs. the vehicle control.

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of IL-8 and IL-10 by LRRC8A Inhibition through the NOX2–Nrf2–CEBPB Transcriptional Axis in THP-1-Derived M 2 Macrophages

doi: 10.3390/ijms25179612

Figure Lengend Snippet: HDAC3-mediated transcriptional repression of LRRC8A in M 2 -MACs. ( A – C ) Real-time PCR examination of LRRC8A ( A ), NSUN2 ( B ), and YBX1 ( C ) in native THP-1 and M 0 - and M 2 -MACs ( n = 4 for each). Expression levels are shown as the ratio to the ACTB. ( D , E ) Effects of the siRNA-mediated NSUN2 inhibition (siNSUN2) ( D ) and pharmacological inhibition of YBX-1 with SU056 (10 μM) for 12 h ( E ) on the expression levels of LRRC8A transcripts in M 2 -MACs ( n = 4 for each). ( F , G ) Identification of histone deacetylase (HDAC) ( F ) and sirtuin (SIRT) ( G ) isoforms expressed in M 2 -MACs by a real-time PCR examination. Expression levels are shown as the ratio to the ACTB ( n = 4 for each). ( H ) Effects of treatments with the pan-HDAC inhibitor, vorinostat (1 μM), the HDAC1/2 dual inhibitor, AATB (10 μM), the selective HDAC3 inhibitor, T247 (10 μM), and the selective SIRT1 inhibitor, Ex527 (1 μM), for 24 h on the expression levels of LRRC8A transcripts in M 2 -MACs. ( I ) Effects of siRNA-mediated HDAC3 inhibition (siHDAC3) on the LRRC8A expression levels in M 2 -MACs. mRNA expression levels in siCont and the vehicle control are expressed as 1.0 ( n = 4 for each). **: p < 0.01 vs. the vehicle control or siCont.

Article Snippet: IL-10/IL-8 human uncoated enzyme-linked immunosorbent assay (ELISA) kits (88-7106-77, 88-8086-77), the Halt TM phosphatase inhibitor cocktail (SF-78420), and validated/pre-designed siRNAs for LRRC8A (siRNA ID: s32107, pre-designed), Nrf2 (s9491, pre-designed), VDAC1 (s14768, validated), CLIC1 (s636, validated), ANO6 (s225825, pre-designed), CLCN3 (s3136, validated), CEBPB (s2892, pre-designed), NSUN2 (s29683, pre-designed), and WNK1 (s35234, validated) were from Thermo Fisher Scientific (Waltham, MA USA). siRNAs for control-A (sc-37007) and HDAC3 (sc-35538) were from Santa Cruz Biotechnology, Sant Cruz, CA, USA.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Inhibition, Histone Deacetylase Assay, Control