c/ebpβ-2 c/ebpβ-3  (Addgene inc)

Journal: bioRxiv

Article Title: Oncogenic functions of the m6A demethylase FTO in breast cancer cells involving translational upregulation of C/EBPβ-LIP

doi: 10.1101/2023.09.21.558784

Figure Lengend Snippet: A ) Schematic overview of the human CEBPB mRNA with indicated translation start sites for LAP1, LAP2, LIP and the uORF. Predicted (SRAMP, ) and experimentally detected m6A modifications from HEK cells (miCLIP: Linder et al. 2015 , DARTseq Meyer 2019 ) are indicated with adenosine base number. B) Immunoblot showing reduced levels of C/EBPβ-LIP upon knockdown of FTO (shFTO) in MDA-MB-231 cells versus scrambled control (SCR). β-actin was used as loading control. C ) Decay curves of CEBPB mRNA from MDA-MB-231 cells with FTO knockdown (shFTO1 or shFTO2) versus shRNA-scrambled (scr) control. Values represent mean ± S.E.M. of 3 biological replicates. Significance was determined by multiple Student’s T-test with Holm-Sidak correction. D ) Immunoblot showing increased levels of C/EBPβ-LIP upon knockdown of WTAP (shWTAP) in MDA-MB-231 and BT-20 cells versus scrambled control (SCR). β-actin was used as loading control. E ) Quantification of C/EBPβ-LIP/LAP ratio in MDA-MB-231 or BT-20 cells with WTAP knockdown. Significance was determined by Student’s T-test. **: p<0.01, ***: p<0.001 (n=3 biological replicates).

Article Snippet: The following primary antibodies were used for detection: FTO: NB110-60935, Novus Biologicals (1:1666); β-Actin: 691001, MP Bio (1:5000); C/EBPβ: ab32358, Abcam (1:1000); WTAP: #56501 (1:1000), Cell Signaling; COL1A1: #72026 (1:1000), Cell Signaling; SMAD6: NB100-56440, Novus Biologicals (1:1000).

Techniques: Western Blot, shRNA

Journal: bioRxiv

Article Title: Oncogenic functions of the m6A demethylase FTO in breast cancer cells involving translational upregulation of C/EBPβ-LIP

doi: 10.1101/2023.09.21.558784

Figure Lengend Snippet: A ) Transwell migration assay of MDA-MB-231 cells with FTO-knockdown (shFTO1) or scrambled control (scr) with overexpression C/EBPβ-LIP or empty vector (EV) control, at 48 hours. The graph shows quantification of migrated cells with pictures of stained cells presented at the bottom. Scale bar represents 500 μm. Significance determined by one way ANOVA with Dunnett’s posthoc test, **: p<0.01 (n=4 technical replicates). B ) Immunoblot showing overexpression of C/EBPβ-LIP in FTO knockdown (shFTO1 or shFTO2) or scrambled-shRNA (scr) control MDA-MB-231 cells. β-actin was used as loading control.

Article Snippet: The following primary antibodies were used for detection: FTO: NB110-60935, Novus Biologicals (1:1666); β-Actin: 691001, MP Bio (1:5000); C/EBPβ: ab32358, Abcam (1:1000); WTAP: #56501 (1:1000), Cell Signaling; COL1A1: #72026 (1:1000), Cell Signaling; SMAD6: NB100-56440, Novus Biologicals (1:1000).

Techniques: Transwell Migration Assay, Over Expression, Plasmid Preparation, Staining, Western Blot, shRNA

Journal: bioRxiv

Article Title: Oncogenic functions of the m6A demethylase FTO in breast cancer cells involving translational upregulation of C/EBPβ-LIP

doi: 10.1101/2023.09.21.558784

Figure Lengend Snippet: A ) Second transwell migration assay of MDA-MB-231 cells with FTO knockdown (shFTO1) or scrambled control (scr) with overexpression C/EBPβ-LIP or empty vector (EV) control, at 48 hours. The graph shows quantification of migrated cells with pictures of stained cells presented at the bottom. Scale bar represents 500 μm. Significance determined by one way ANOVA with Dunnett’s posthoc test, **: p<0.01 (n=4 technical replicates). B ) Immunoblot showing overexpression of C/EBPβ-LIP in FTO-knockdown (shFTO1 or shFTO2) or scrambled-

Article Snippet: The following primary antibodies were used for detection: FTO: NB110-60935, Novus Biologicals (1:1666); β-Actin: 691001, MP Bio (1:5000); C/EBPβ: ab32358, Abcam (1:1000); WTAP: #56501 (1:1000), Cell Signaling; COL1A1: #72026 (1:1000), Cell Signaling; SMAD6: NB100-56440, Novus Biologicals (1:1000).

Techniques: Transwell Migration Assay, Over Expression, Plasmid Preparation, Staining, Western Blot

Journal: PLoS ONE

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0095087

Figure Lengend Snippet: ( A ) RT-PCR analysis showing increased expression of galectin-7 in human breast cancer cells after transfection with an expression vector encoding C/EBPβ-2. The two lanes represent two different samples. No such increase was observed in cells transfected with an expression vector encoding C/EBPβ-3. Similar results were obtained with HaCaT cells, a keratinocyte cell line which constitutively expresses galectin-7 . An empty pCMV5 vector was used as transfection control (CTRL) and GAPDH was used as loading control. ( B ) RT-PCR analyses showing expression of galectin-7 mRNA levels in MCF-7 cells after transfection with increasing doses of an expression vector encoding C/EBPβ-2. GAPDH was used as loading control. ( C ) RT-PCR analysis of MCF-7 cells co-transfected with vectors encoding C/EBPβ-2 and C/EBPβ-3. Below, control Western blot analysis showing expression of C/EBPβ-2 and C/EBPβ-3 after transfection. β-actin was used as loading control.

Article Snippet: Vectors encoding human C/EBPα or β (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/EBPβ-2 and C/EBPβ-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0095087

Figure Lengend Snippet: MCF-7 or MDA-MB-231 cells were transfected with an expression vector encoding C/EBPβ-2 before cell fixation and permeabilization. A goat anti-human galectin-7 polyclonal antibody was used in combination with an Alexa Fluor 488-conjugated donkey anti-goat IgG to detect endogenous galectin-7 (green). Nuclei were stained with DAPI (blue).

Article Snippet: Vectors encoding human C/EBPα or β (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/EBPβ-2 and C/EBPβ-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially.

Techniques: Transfection, Expressing, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0095087

Figure Lengend Snippet: ( A ) Schematic representation of C/EBP binding sites within the 5′ flanking region of the human galectin-7 gene. A s eries of 5′ deletion constructs of the 1500 bp galectin-7 promoter region was generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were transfected in MCF-7 and MDA-MB-468 cells. Locations of the putative C/EBP binding sites in the promoter, as determined using the TFsearch computational tool, are shown as empty boxes. ( B ) Sequence analysis of the C/EBP binding sites located at positions -105-98 and the -145-140. ( C ) Mutated constructs of the C/EBPβ binding sites on the 200 bp galectin-7 promoter region were generated and cloned into the pGL3 Basic luciferase reporter vector. The resulting plasmids were co-transfected in MCF-7 cells and were compared to the wild-type p200-galectin-7 promoter. Transfection efficiency was normalized by co-transfection with a β-galactosidase reporter vector.

Article Snippet: Vectors encoding human C/EBPα or β (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/EBPβ-2 and C/EBPβ-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially.

Techniques: Binding Assay, Construct, Generated, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Sequencing, Cotransfection

Journal: PLoS ONE

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0095087

Figure Lengend Snippet: Immunohistological analysis of galectine-7 and C/EBPβ expression in ( A ) human normal epithelial tissues and ( B ) in normal mammary gland tissue and breast carcinoma. Data were provided by the human protein atlas database ( http://www.proteinatlas.org/ ) and by .

Article Snippet: Vectors encoding human C/EBPα or β (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/EBPβ-2 and C/EBPβ-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially.

Techniques: Expressing

Journal: PLoS ONE

Article Title: The CCAAT/Enhancer-Binding Protein Beta-2 Isoform (CEBPβ-2) Upregulates Galectin-7 Expression in Human Breast Cancer Cells

doi: 10.1371/journal.pone.0095087

Figure Lengend Snippet: Data obtained from the Oncomine cancer microarray database ( www.oncomine.org ) showing higher C/EBPβ expression in ( A ) estrogen receptor (ER)-negative and ( B ) triple negative (TN) human breast carcinomas.

Article Snippet: Vectors encoding human C/EBPα or β (SC303472 and SC319561; Origene, Burlington, ON) and vectors encoding C/EBPβ-2 and C/EBPβ-3 (No. 15738 and No. 15737; Addgene, Cambridge, MA) were obtained commercially.

Techniques: Microarray, Expressing

Journal: BMC Cancer

Article Title: NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells

doi: 10.1186/1471-2407-13-570

Figure Lengend Snippet: Selective E 2 -dependent recruitment of NCOA3 to the endogenous PLAC1 promoter in ERα positive MCF-7 cells. (A) Chromatin immunoprecipitation (ChIP) was performed with chromatin prepared from MCF-7 and SK-BR-3 cells which were either untreated (C), treated with 17-β-estradiol (E 2 ) alone, with ICI alone or with both compounds (E 2 /ICI). The promoter region of PLAC1 containing the C/EBPβ and SP1 elements (−348/-198) and a region upstream of the promoter (−1219/-1064) as negative control were analyzed by PCR following immunoprecipitation with the indicated antibodies. Amplification products from soluble chromatin prior to precipitation are shown as control (Input). (B) Quantitative analysis of the recruitment and occupancy shown in (A) determined by real-time RT-PCR. The results corrected by input are shown as fold increase compared to unstimulated cells used as a reference.

Article Snippet: The purified DNA was analyzed by conventional PCR and quantitative real time PCR for the presence of the human PLAC1 promoter fragment −348/-198 bp containing the C/EBPβ and SP1 elements (sense 5′-CAA CAG CAA GCA CTA CAA GTG-3′; antisense 5′-GAA GCT CAA CTC GGT GCA CTT GTT C-3′) and for a fragment −1219/-1064 bp upstream of the promoter as negative control (sense 5′-AAG CAC TTA GGA CAG CAT CTG-3′; antisense 5′-TGA ATG ATA CCT ACT GTC ATG) following immunoprecipitation with antibodies reactive to SP1 (ab13370), C/EBPβ-2 (ab32358), ERα (ab2746), NCOA1 (ab84), NCOA2 (ab9261), NCOA3 (ab2782), p300 (ab14984), pCAF (ab12188), AcH3 (ab12179), AcH4 (ab15823), TFIIB (ab12094) and RNA-Polymerase II (ab5131) (all from Abcam).

Techniques: Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation, Amplification, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Differential Expression and Function of Stamp Family Proteins in Adipocyte Differentiation

doi: 10.1371/journal.pone.0068249

Figure Lengend Snippet: (A) Western analysis showing C/ebpβ protein levels at day 0 (d0), day 1 (d1) and day 2 (d2) of differentiation with the same cells as in (5A). (B) Quantification of Western analysis results in (A) with relative C/ebpβ protein levels normalized to β-actin from two independent experiments, n = 6. #p<0.05 between brackets.

Article Snippet: Cells were then blocked with 1% BSA (Sigma-Aldrich) for 30 min before incubation with C/ebpβ antiserum (1∶100) (Abcam, ab32358) at 4°C overnight and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibodies (1∶500) (Invitrogen) for 1 h at room temperature.

Techniques: Western Blot

Journal: Journal of Translational Medicine

Article Title: Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

doi: 10.1186/s12967-022-03779-9

Figure Lengend Snippet: C/EBPβ is a transcription activator in the promoter region of Clec7a gene. ( A and B ) Schematic representation of a series of 5’ unidirectional deletions of the 2000 bp Clec7a promoter region fused in frame to pcDNA3.1 luciferase reporter vector, and then the promoter activity of C/EBPβ was determined by measuring the fluorescence intensity. C/EBPβ binding elements in the promoter region of Clec7a were predicted using the transcription activator search software, and shown as cyan boxes ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 compared with pcDNA3.1-GFP-NC group via unpaired Student’s t test. ( C ) Mutated C/EBPβ binding sites on Clec7a promoter-driven luciferase reporters were constructed, and then the luciferase activity was analyzed via one-way ANOVA followed by Tukey’s post hoc test ( n = 3). *** P < 0.001 compared with the pcDNA3.1-GFP-NC + psiPRO-Clec7a-p500 group. ### P < 0.001 compared with the pcDNA3.1-GFP-NC + psiPRO-Clec7a-p500-DEL group. D The location of amplified fragments of three pairs of primers on Clec7a promoter region. E Chromatin immunoprecipitation (ChIP) analysis of C/EBPβ binding at the Clec7a promoter. The anti-C/EBPβ antibody or IgG control was used to pull down the DNA–protein complex from the control or LPS + ATP treated BV2 cells as indicated. Subsequently, immunoprecipitated DNA was determined by PCR and quantitative real-time PCR with three pairs of primers ( n = 3). Data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test was used to analyze data among multiple groups. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the BV2-C/EBPβ antibody group

Article Snippet: The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃.

Techniques: Luciferase, Plasmid Preparation, Activity Assay, Fluorescence, Binding Assay, Software, Construct, Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Journal of Translational Medicine

Article Title: Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

doi: 10.1186/s12967-022-03779-9

Figure Lengend Snippet: C/EBPβ knockdown attenuated neuropathic pain by regulating Clec7a-NLRP3 induced pyroptosis in vivo. A Timeline schematic of experimental paradigm. B The decreased expression of C/EBPβ significantly ameliorated mechanical withdrawal threshold in CCI rat model ( n = 5–10 rats/group). C Intrathecally administration of Cebpb siRNA significantly reduced the expression of C/EBPβ, Clec7a, pyroptosis-related proteins (NLRP3, GSDMD) and pSyk, pERK, pJNK in ipsilateral spinal cord of CCI rats on postoperative day 10 ( n = 3 independent blots). D Immunohistochemical detection of C/EBPβ and Clec7a in ipsilateral SC of CCI rats after si- Cebpb administration ( n = 6 rats/group, 1 section/rat). White arrows point to C/EBPβ- or Clec7a-positive cells, respectively. Scale bar: 100 μm (20 ×); 50 μm (40 ×). Data are presented as mean ± SEM. Two-way repeated measures ANOVA and Tukey’s post hoc test was used to analyze data at different time points B . One-way ANOVA was used to analyze data among multiple groups followed by Tukey’s post hoc test for equal variances or Dunnett T3 post hoc test for unequal variances C – D . * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the Sham group; ## P < 0.01, ### P < 0.001 compared with sham + si-NC group; △ P < 0.05, △△ P < 0.01, △△△ P < 0.001 compared with CCI + si-NC group

Article Snippet: The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃.

Techniques: In Vivo, Expressing, Immunohistochemical staining

Journal: Journal of Translational Medicine

Article Title: Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

doi: 10.1186/s12967-022-03779-9

Figure Lengend Snippet: C/EBPβ knockdown inhibited the overexpression of Clec7a and NLRP3 induced pyroptosis in spinal cord of the chronic constriction injury rats. A The double immunofluorescence staining of C/EBPβ and Clec7a in ipsilateral SC of CCI rats after si-Cebpb administration (n = 6 rats/group, 1 section/rat). White arrows point to C/EBPβ- and Clec7a-positive cells. Scale bar: 50 μm. B Representative images (left) and quantification evaluation (right) of Tunel staining in the indicated groups ( n = 6 rats/group, 1 section/rat). Red: Tunel-positive cells; blue: nuclei (Hoechst 33,258). Scale bar: 100 μm. Data are presented as mean ± SEM. One-way ANOVA was used to analyze data among multiple groups followed by Tukey’s post hoc test for equal variances or Dunnett T3 post hoc test for unequal variances. * P < 0.05, ** P < 0.01 compared with the Sham group; ## P < 0.01 compared with sham + si-NC group; △ P < 0.05, △△ P < 0.01 compared with CCI + si-NC group

Article Snippet: The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃.

Techniques: Over Expression, Double Immunofluorescence Staining, TUNEL Assay, Staining

Journal: Journal of Translational Medicine

Article Title: Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

doi: 10.1186/s12967-022-03779-9

Figure Lengend Snippet: Disruption of the C/EBPβ-Clec7a axis attenuated neuropathic pain by regulating NLRP3 inflammasome-induced pyroptosis in vivo. A Timeline schematic of experimental paradigm. B The decreased expression of C/EBPβ significantly ameliorated mechanical withdrawal threshold in CCI rat model, while overexpression Clec7a facilitated neuropathic pain (Sham group, n = 9; the other groups, n = 15 rats/group). C Expression levels of C/EBPβ, Clec7a, pSyk, pERK, pJNK, NLRP3 and GSDMD proteins in ipsilateral spinal cord (SC) of CCI rats on postoperative 4 weeks were measured using western blot analysis ( n = 3 independent blots). D Immunohistochemical detection of C/EBPβ and Clec7a in ipsilateral SC of CCI rats after treatment ( n = 9 rats/group, 1 section/rat). White arrows point to C/EBPβ- or Clec7a-positive cells, respectively. Scale bar: 100 μm (20 ×); 50 μm (40 ×). E The concentration of IL-1β and IL-18 in serum of indicated groups were assessed by ELISA ( n = 3 rats/group with 3 technical replicates). F Representative images (up) and quantification evaluation (down) of Tunel staining in the indicated groups ( n = 6 rats/group, 3 section/rat). Red: Tunel-positive cells; blue: nuclei (Hoechst 33,258). Scale bar: 100 μm. Data are presented as mean ± SEM. Two-way repeated measures ANOVA and Tukey’s post hoc test was used to analyze data at different time points B . One-way ANOVA was used to analyze data among multiple groups followed by Tukey’s post hoc test for equal variances or Dunnett T3 post hoc test for unequal variances C – F . ** P < 0.01, *** P < 0.001 compared with the Sham group; # P < 0.05, ## P < 0.01, ### P < 0.001 compared with CCI + pLV.1-NC + pLVSO5-NC group; △ P < 0.05, △△ P < 0.01, △△△ P < 0.001 compared with CCI + pLV.1-sh Cebpb + pLVSO5-NC group; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 compared with CCI + pLV.1-NC + pLVSO5- Clec7a- overexpress group

Article Snippet: The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃.

Techniques: In Vivo, Expressing, Over Expression, Western Blot, Immunohistochemical staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining

Journal: Journal of Translational Medicine

Article Title: Disruption of C/EBPβ-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain

doi: 10.1186/s12967-022-03779-9

Figure Lengend Snippet: Illustration on the critical role of C/EBPβ-Clec7a axis in neuropathic pain progression, which supports the idea that CCI-induced overexpression of Clec7a, and the direct interaction between C/EBPβ and Clec7a observed along the pain pathways may contribute to neuropathic pain by triggering the phosphorylation of Syk, ERK and JNK proteins, as well as activating NLRP3 inflammasome-mediated pyroptosis

Article Snippet: The chromatin was then immunoprecipitated with 2 μg (0.6 μg/μL, 3.4 μL) of rabbit anti-C/EBPβ antibody (ab32358, Abcam, Cambridge, MA, USA) or 2 μg of rabbit IgG with rotation overnight at 4 ℃.

Techniques: Over Expression