anti klf5  (Proteintech)

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: PKA signaling pathway aberrantly activated in PDAC (A) Heatmap showing mRNA expression levels of moderately and poorly differentiated PDAC from the GEO database ( GSE226307 ). PRKAR2B and KLF5 were labeled. (B) KEGG pathway enrichment analysis of differentially expressed genes from RNA-seq in GEO database ( GSE226307 ). The “cAMP signaling pathway” is highlighted in red. (C) Volcano plot of RNA-seq data comparing PDAC tissue and normal pancreatic tissue ( GSE16515 ). Red dots represent upregulated mRNAs, blue dots represent downregulated mRNAs ( p < 0.05, log 2 foldchange >1 or < –1). PRKAR2B and KLF5 were labeled. (D–F) Colorimetric analysis of PKA activity (D) and ELISA analysis of pCREB and KLF5 levels (E–F) in pancreatic puncture fluid from patients with benign pancreatic diseases ( n = 3 independent samples), well or moderately differentiated PDAC ( n = 10 independent samples), or poorly differentiated PDAC ( n = 7 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01. (G and H) Colorimetric analysis of PKA activity (G) and qPCR analysis of mRNA expression of KLF5 (H) in H6C7, Capan-2, and AsPC-1 cells ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01, ∗∗∗, p < 0.001. (I and J) mRNA expression profiles of KLF5 based on data from 182 PDAC patients and 4 normal controls in the TCGA database. ∗, p < 0.05. (K) Kaplan-Meier survival analysis of KLF5 expression using data from 182 PDAC patients in the TCGA database. ∗, p < 0.05.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: Expressing, Labeling, RNA Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: PRKAR2B downregulation activates PKA in PDAC (A) Volcano plot of RNA-seq data comparing PDAC tissue and normal pancreatic tissue ( GSE226307 ). Red dots represent upregulated mRNAs, blue dots represent downregulated mRNAs ( p < 0.05, log 2 foldchange >1 or < -1). KLF5, PRKAR1A, PRKAR1B, PRKAR2A, PRKAR2B, PRKACA, and PRKACB were labeled. (B) mRNA expression levels of PRKAR2B based on data from 182 PDAC patients and 4 normal controls in the TCGA database. ∗, p < 0.05. (C) Protein expression levels of PRKAR2B based on data from 137 PDAC patients and 74 normal controls in the CPTAC database. ∗∗∗∗, p < 0.0001. (D) Kaplan-Meier survival analysis of PRKAR2B expression using data from 182 PDAC patients in the TCGA database. ∗, p < 0.05. (E) mRNA expression levels of indicated genes in AsPC-1 cells with or without siRNA ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001. (F) Colorimetric analysis of PKA activity in AsPC-1 cells transfected with siRNA targeting EPHA2 , SEMA4B , PYGB , or PRKAR2B ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01; NS, no significance. (G) Schematic diagram of PKA activation and inactivation. RS, regulatory subunit; CS, catalytic subunit. (H) Immunoblot analysis of PRKAR2B in H6C7, Capan-2, and AsPC-1 cells. (I) Immunoblot analysis of PRKAR2B in AsPC-1 cells with PRKAR2B knockout or overexpression. (J) Colorimetric analysis of PKA activity in AsPC-1 cells with PRKAR2B knockout or overexpression ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05. (K) Immunoblot analysis of CREB, pCREB, and KLF5 in AsPC-1 cells with PRKAR2B knockout or overexpression. (L) Correlation between PRKAR2B and KLF5 expression based on data from 182 PDAC patients in the TCGA database. ∗∗∗∗, p < 0.0001.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Labeling, Expressing, Activity Assay, Transfection, Activation Assay, Western Blot, Knock-Out, Over Expression

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: HHEX downregulation represses PRKAR2B transcription (A) Enrichment of H3K27me3 at the PRKAR2B gene in AsPC-1 cells. Chromatin immunoprecipitation was performed using an antibody against H3K27me3 or a control IgG antibody. The enriched DNA was quantified by qPCR and is presented as % of Input. Values are shown as mean ± SD ( n = 3 independent repeats). NS, no significance. (B and C) Overlap of the upregulated (B) and downregulated (C) genes in moderate and poor differentiation PDAC compared with normal pancreatic tissue with known transcription factor. (D) mRNA expression profile of HHEX using 182 PDAC patient data and 4 normal controls from TCGA database. ∗∗, p < 0.01. (E) Kaplan-Meier survival analysis of HHEX expression using data from 182 PDAC patients in the TCGA database. ∗∗, p < 0.01. (F) Correlation between PRKAR2B and HHEX expression based on data from 182 PDAC patients in the TCGA database. ∗∗∗, p < 0.001. (G) Enrichment of HHEX at the PRKAR2B gene in AsPC-1 cells. Chromatin immunoprecipitation was performed using an antibody against HHEX or a control IgG antibody. The enriched DNA was quantified by qPCR and is presented as % of input. Values are shown as mean ± SD ( n = 3 independent repeats). ∗∗∗∗, p < 0.0001. (H and I) mRNA levels of HHEX (H) or PRKAR2B (I) in AsPC-1 cells with HHEX knockdown or overexpression ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01, ∗∗∗, p < 0.001. (J) mRNA levels of HHEX in AsPC-1 cells with PRKAR2B knockout or overexpression ( n = 3 independent repeats). Data were expressed as mean ± SD. NS, no significance. (K–L) Colorimetric analysis of PKA activity (K) and qPCR analysis of KLF5 mRNA levels (L) in AsPC-1 cells with or without PRKAR2B knockout followed by HHEX knockdown ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗, p < 0.01, ∗∗∗∗, p < 0.0001; NS, no significance.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: Chromatin Immunoprecipitation, Control, Expressing, Knockdown, Over Expression, Knock-Out, Activity Assay

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: PKA activation promotes PDAC progression through metabolic reprogramming (A and B) Colorimetric analysis of PKA activity (A) and qPCR analysis of KLF5 mRNA levels (B) in AsPC-1 cells treated with or without KT5720 (5 μM, 48 h) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗∗, p < 0.001. (C) Immunoblot analysis of CREB, pCREB, and KLF5 in AsPC-1 cells following PRKAR2B knockout or overexpression, with or without KT5720 treatment (5 μM, 48 h). (D) Cell viability was measured by CCK-8 assay in AsPC-1 cells subjected to PRKAR2B knockout or HHEX knockdown, in the presence or absence of treatment with KT5720 (5 μM, 48 h) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗∗, p < 0.001; NS, no significance. (E) Cell death analysis by PI staining and flow cytometry in AsPC-1 cells subjected to PRKAR2B knockout or HHEX knockdown, in the presence or absence of treatment with KT5720 (5 μM, 48 h) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01; NS, no significance. (F) Cell invasion assays in AsPC-1 cells following PRKAR2B knockout or HHEX knockdown, with or without KT5720 treatment (5 μM, 48 h). Scale bars, 80 μm (G) KEGG pathway enrichment analysis of differentially expressed genes identified in GEO database ( GSE134618 ). (H) Cell viability measured by CCK-8 assay in AsPC-1 cells under low FBS (2%) or low glucose (1,000 mg/L) conditions, with or without KT5720 treatment (5 μM, 48 h, n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; NS, no significance. (I) Cell death analysis by PI staining and flow cytometry in AsPC-1 cells under low FBS (2%) or low glucose (1,000 mg/L) conditions, with or without KT5720 treatment (5 μM, 48 h, n = 3 independent repeats). Data were expressed as mean ± SD. NS, no significance. (J) Cell invasion assays in AsPC-1 cells treated with low FBS (2%), low glucose (1,000 mg/L), or KT5720 (5 μM, 48 h). Scale bars, 80 μm (K) Relative lactate levels in pancreatic puncture fluid from patients with benign pancreatic diseases ( n = 3 independent samples), well or moderately differentiated PDAC ( n = 10 independent samples), or poorly differentiated PDAC ( n = 7 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01. (L) Relative lactate levels in the supernatant of H6C7, Capan-2, and AsPC-1 cells ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05. (M) Intracellular and extracellular glucose levels in H6C7, Capan-2, and AsPC-1 cells ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: Activation Assay, Activity Assay, Western Blot, Knock-Out, Over Expression, CCK-8 Assay, Knockdown, Staining, Flow Cytometry

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: Glucose-activated PKA promotes PDAC progression via HK2-driven glycolysis (A) Colorimetric analysis of PKA activity in AsPC-1 cells treated with high glucose (4,500 mg/L) or low glucose (1,000 mg/L) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05. (B) Immunoblot analysis of CREB, pCREB, and KLF5 in AsPC-1 cells treated with high glucose (4,500 mg/L) or low glucose (1,000 mg/L). (C–E) mRNA expression of KLF5 (C), intracellular cAMP levels (D), and relative ATP levels (E) in AsPC-1 cells treated with high glucose (4,500 mg/L) or low glucose (1,000 mg/L) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗, p < 0.01. (F) Schematic diagram of carbohydrate metabolism. TCA, tricarboxylic acid; PPP, pentose phosphate pathway. (G) Intracellular and extracellular glucose levels in AsPC-1 cells with or without PRKAR2B knockout ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗∗, p < 0.001. (H) Relative lactate levels in the supernatant of AsPC-1 cultures with or without PRKAR2B knockout ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01. (I–J) NADP + /NADPH ratio (I) and relative ATP levels (J) in AsPC-1 cells with or without PRKAR2B knockout ( n = 3 independent repeats). Data were expressed as mean ± SD. NS, no significance. (K) Schematic diagram of the 19 core genes identified in the network analysis of C. (L) mRNA expression levels of HK2 based on data from 182 PDAC patients and 4 normal controls in the TCGA database. ∗∗∗∗, p < 0.0001. (M) Protein expression levels of HK2 based on data from 137 PDAC patients and 74 normal controls in the CPTAC database. ∗∗∗∗, p < 0.0001. (N–O) Correlation analysis among HK2 , KLF5 , and PRKAR2B expression using transcriptomic data from 182 PDAC patients in TCGA. ∗∗∗, p < 0.001, ∗∗∗∗, p < 0.0001. (P) HK2 mRNA expression in AsPC-1 cells following PRKAR2B knockout or treatment with dbcAMP (0.2 mM, 24 h) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01. (Q) HK2 mRNA expression in AsPC-1 cells with or without HK2 knockdown ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗, p < 0.05. (R) Cell viability measured by CCK-8 assay in AsPC-1 cells after HK2 knockdown or treatment with 2-DG (4 mM, 24 h) ( n = 3 independent repeats). Data were expressed as mean ± SD. ∗∗, p < 0.01; NS, no significance. (S) Cell invasion assays in AsPC-1 cells after HK2 knockdown or treatment with 2-DG (4 mM, 24 h). Data were expressed as mean ± SD. Scale bars, 80 μm.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: Activity Assay, Western Blot, Expressing, Knock-Out, Knockdown, CCK-8 Assay

Journal: iScience

Article Title: HHEX-PRKAR2B axis-mediated PKA activation drives glucose metabolism-dependent progression of pancreatic ductal adenocarcinoma

doi: 10.1016/j.isci.2026.114691

Figure Lengend Snippet: In vivo demonstration of glucose-activated PKA promoting glycolysis-dependent progression A. Immunoblot analysis of PRKAR2B in KPC cells with or without PRKAR2B knockout. (B–D) Representative images of colony formation by KPC cells, with or without PRKAR2B knockout, cultured in normal or low-glucose medium (B). Colony size (C) and number of colonies (D) were graphed ( n = 3 independent repeats). Data were expressed as mean ± SD. Scale bars, 50 μm ∗, p < 0.05, ∗∗, p < 0.01, NS, no significance. (E) Representative H&E-stained pancreatic sections from mice with tamoxifen-inducible, pancreas-specific KRAS expression, harvested 3 weeks after tamoxifen or vehicle injection. Scale bars, 1 mm (F–H) PKA activity (F), pyruvic acid level (G), and lactate level (H) in pancreatic tissues from mice, harvested 3 weeks after tamoxifen or vehicle injection, assessing the effect of tamoxifen-inducible, pancreas-specific KRAS expression ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01. (I) Colorimetric analysis of PKA activity in wild-type and diabetic mice ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (J) Representative immunoblot of CREB, pCREB, and KLF5 in wild-type and diabetic mice ( n = 4 independent samples; see F for all replicates). (K) mRNA expression level of HK2 in wild-type and diabetic mice ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (L) Schematic diagram of the experimental design in mice. Pan02 cells with or without PRKAR2B knockout was injected into wild type or diabetic mouse through the tail vein ( n = 4 independent samples). (M) Changes in mouse body weight following tail vein injection of PRKAR2B -knockout or wild-type Pan02 cells ( n = 4 independent samples). Data were expressed as mean ± SD. ∗∗, p < 0.01. (N) Colorimetric analysis of PKA activity in tumor allografts from wild-type and diabetic mice with or without PRKAR2B knockout ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05, ∗∗, p < 0.01. (O) Representative image of Ki67 IHC staining in Pan02-control and Pan02- PRKAR2B -sg allografts from wild-type or diabetic mice ( n = 4 independent samples). Scale bars, 150 μm (P) Representative H&E-stained lung sections at the experimental endpoint. Scale bars, 3 mm (Q–R) Quantification of metastatic lesion number (Q) and total metastatic burden (R) at the experimental endpoint across the indicated treatment groups ( n = 4 independent samples). Data were expressed as mean ± SD. ∗, p < 0.05; NS, no significance.

Article Snippet: The following primary antibodies were used: GAPDH (1:1000, cat. sc-137179, Santa Cruz Biotechnology), PRKAR2B (1:1000, cat. sc-376778, Santa Cruz Biotechnology), CREB (1:1000, cat. ab31515, Abcam), pCREB (1:1000, cat. ab32096, Abcam), KLF5 (1:1000, cat. sc-398014, Santa Cruz Biotechnology), and HK2 (1:1000, cat. sc-130358, Santa Cruz Biotechnology).

Techniques: In Vivo, Western Blot, Knock-Out, Cell Culture, Staining, Expressing, Injection, Activity Assay, Immunohistochemistry, Control

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: Identification of co-regulators for NR5A2. (A) Co-enrichment of TF motifs with NR5A2 peaks in each cell stage. The color indicates the odds ratio between observed and expected motif occurrence within the peak. The size of circle indicates the P -value. (B) Ribo-seq data showing the expression levels of KLF and GATA families at different developmental stages. CUT&Tag profiles for KLF5 and GATA6 with two replicates at the 8-cell and morula stages are shown. (D) Venn diagrams showing the number of overlapping peaks between NR5A2, KLF5 and GATA6 at the 8-cell and the morula stages. (E) Heatmap showing the enrichment of NR5A2, KLF5, GATA6, ATAC-seq and H3K27ac from the 2-cell to the morula stage with four different clusters. SINE B1/Alu motif occurrence is shown on the right. All maps extend for 2 kb on either side. 4C, 4-cell stage; 8C, 8-cell stage; E2C, early 2-cell embryo; FGO, full-grown oocytes; ICM, inner cell mass; L2C, late 2-cell embryo; LPI, late prometaphase I oocytes; MII, metaphase II oocytes; PN, pronuclear stage. (C) IGV snapshot showing co-occupancy of KLF5 (green) and GATA6 (blue) with NR5A2 (red).

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: NR5A2 and KLF5 contribution to pre-implantation development. (A) Schematic of siRNA-mediated knockdown. siRNA targeting Nr5a2 and/or Klf5 was microinjected into zygotes. Immunostaining and genomics assays were performed with 8-cell embryos. (B) Abundance of co-injection marker H2B-EGFP , Nr5a2 and Klf5 transcripts in control (blue), Nr5a2 KD (pink), Klf5 KD (yellow) and Nr5a2 Klf5 dKD (purple) 8-cell embryos. P -values ( t -test, two-sided) are shown. Each dot represents a replicate of the RNA-seq experiment. Error bars represent s.d. (C,D) Representative images (C) and quantification (D) of immunostaining analysis showing NR5A2 (magenta), KLF5 (green) and DAPI (cyan) staining in 8-cell embryos. NR5A2 and KLF5 signals are shown in mid-section images. DAPI signals are presented as full z -stack images to visualize all nuclei. The number of embryos examined ( n ) from three independent experiments is indicated. Scale bars: 20 µm. Bars overlaid on the plots indicate means. P -values ( t -test, two-sided) are shown. (E) Stereomicroscopic representative images showing embryonic development at different time points under different KD conditions (top to bottom: control, Nr5a2 KD, Klf5 KD, dKD). Scale bars: 75 µm. (F) Quantification of embryonic development experiments. The number of embryos examined ( n ) from three or four independent experiments were as follows: control, n =10, 8, 24 and 20; Nr5a2 KD, n =10, 19 and 17; Klf5 KD, n =10, 19 and 19; Nr5a2 Klf5 dKD, n =14, 15 and 16.

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Knockdown, Immunostaining, Injection, Marker, Control, RNA Sequencing, Staining

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: Transcriptional regulation by NR5A2 and KLF5 at the 8-cell stage. (A) MA plots of the log 2 fold-change ( Nr5a2 KD/control, dKD/control and Klf5 KD/control) in gene expression in Nr5a2 KD (left), dKD (center) and Klf5 KD (right). The number of up- and downregulated genes in each condition is shown. (B) Venn diagram showing the number of downregulated genes overlapping in Nr5a2 KD, dKD and Klf5 KD. The dKD-specific downregulated category contains genes related to Wnt/TGFβ signaling ( Prkcd , Smad7 and Ryk ) and DNA repair ( Rad51c , Rnaseh2c , Pold1 and Aunip ). (C) Differential binding analysis of H3K27ac CUT&Tag in Nr5a2 KD (left), dKD (center) and Klf5 KD (right). The numbers indicate gain and loss of H3K27ac regions in each condition. (D) Heatmap showing enrichment of H3K27ac on gain and loss of H3K27ac regions identified by differential binding analysis. H3K27 CUT&Tag data from three biological replicates merged are shown in each condition. (E) IGV snapshot highlighting the representative early ICM, TE, Hippo signaling pathway genes, and dKD-specific downregulated genes. H3K27ac CUT&Tag data are shown as merged profiles. (F) PLA showing a close association between NR5A2 and KLF5 in the nucleus. Eight-cell embryos were incubated with NR5A2 and/or KLF5 antibodies, and PLA signals were detected using a confocal microscope. A representative nucleus with z -stack is shown in each condition Scale bars: 10 µm. (G) Quantification of PLA signal density in each condition from two independent experiments (red and cyan). P -values (Mann–Whitney test) are shown. Sample sizes (embryos) in each replicate are as follows: no primary, n =6, 7; anti-NR5A2, n =8, 7; anti-KLF5, n =5, 7; anti-NR5A2+anti-KLF5, n =10, 8.

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Control, Gene Expression, Binding Assay, Incubation, Microscopy, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: Xist expression is regulated by NR5A2 and GATA factors. (A) RNA expression of XCI-related genes and GATA family members. Heatmaps show z -normalized TPM values for each replicate and log 2 fold-changes ( Nr5a2 KD/control, dKD/control and Klf5 KD/control). (B,C) Representative images (B) and quantification (C) of immunostaining analysis showing NR5A2 (magenta), GATA6 (gray) and DAPI (cyan) in 8-cell embryos. NR5A2 and GATA signals are shown in mid-section images. DAPI signals are shown as full z -stack images. The number of embryos examined ( n ) from three independent experiments is indicated. Scale bars: 20 µm. Bars overlaid on the plots indicate means. P -values ( t -test, two-sided) are shown. (D) IGV snapshot showing NR5A2 (red), GATA6 (light blue), GATA1 (dark blue) and KLF5 (green) near the Xist genomic locus. GATA6 and H3K27ac signals from extra-embryonic endoderm (XEN) cells are shown as control. Orange highlighted regions indicate Xist regulatory elements identified in previous studies ( ; ).

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Expressing, RNA Expression, Control, Immunostaining

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: Nucleosome binding by pioneer TFs NR5A2, KLF5 and GATA6. (A) Purified mouse NR5A2 full-length (lane 2), KLF5 DBD (lane 3) and GATA6 DBD (lane 4). Lane 1 shows molecular markers. (B) IGV snapshot showing co-occupancy by NR5A2, KLF5 and GATA6 along with MNase-seq profiles in mESCs. The dashed rectangle highlights the region used for mono-nucleosome reconstitution. 8C, 8-cell stage. (C) The sequence of a 149 bp DNA fragment containing SINE B1 / Alu was used for mono-nucleosome reconstitution. NR5A2 (red), KLF5 (green) and GATA6 (blue) motifs are highlighted. SHL, superhelical location. (D) The cryo-EM structure of B1-nucleosome bound by ScFV. (E,F) Representative gel images (E) and quantification (F) of EMSA. Nucleosomes were analyzed by 5% native-PAGE and detected by Alexa Fluor 647 fluorescence. Data are shown as mean±s.d. for three independent experiments. (G) Representative EMSA gel showing TFs co-binding on the B1-nucleosome. Nucleosomes were analyzed by 6% native-PAGE and detected by Alexa Fluor 647 fluorescence. Reproducibility was confirmed with three independent experiments. (H) Representative EMSA gel showing NKG (NR5A2-KLF5-GATA6) co-binding on the B1-nucleosome. Nucleosomes were analyzed by 6% native-PAGE and detected by Alexa Fluor 647 fluorescence. Reproducibility was confirmed with three independent experiments. (I) Enlarged image of lane 5 from H. An asterisk marks an additional band, potentially non-specific KLF5 DBD binding to the NKG-B1 nucleosome complex.

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Binding Assay, Purification, Sequencing, Cryo-EM Sample Prep, Clear Native PAGE, Fluorescence

Journal: Development (Cambridge, England)

Article Title: Feed-forward loops by NR5A2 ensure robust gene activation during pre-implantation development

doi: 10.1242/dev.205059

Figure Lengend Snippet: Feed-forward loop regulation by NR5A2 during the totipotency-to-pluripotency transition. (A) Model of feed-forward loop mediated by NR5A2. Following zygotic genome activation, NR5A2 regulates a broad set of genes involved in lineage-determining factors, Hippo signaling, chromosome segregation and DNA repair. NR5A2 regulates the expression of KLF and GATA family TFs, which subsequently function as co-regulators of NR5A2. KLF5 and GATA6 co-occupy the chromatin with NR5A2 and further regulate transcription, such as Wnt signaling and Xist , ensuring proper embryonic development. (B) Proposed models of NR5A2 pioneer factor function in the mouse pre-implantation embryos. (i) NR5A2 recognizes and binds to its own motif sequence on nucleosomal DNA. NR5A2 locally opens the closed chromatin and recruits a histone acetyltransferase (HAT), such as p300, to establish active promoters or enhancers. (ii) Following local chromatin opening, both NR5A2 and KLF5 recruit a HAT to enhance histone acetylation for transcriptional activation. GATA6 also potentially binds to nucleosomes and stimulates transcription through its pioneering activity. 2C, 2-cell stage; 4C, 4-cell stage; 8C, 8-cell stage; REs, regulatory elements.

Article Snippet: Embryos were incubated in antibody buffer [20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1× Protease inhibitor cocktail (Roche) with primary antibody [1:100; NR5A2 (R&D Systems, PP-H2325-00), KLF5 (Proteintech, 21017-1-AP), GATA6 (R&D Systems, AF1700), H3K27ac (Active motif, 39133)] overnight at 4°C on nutator.

Techniques: Activation Assay, Expressing, Sequencing, Activity Assay

Journal: Science Advances

Article Title: HERVH-derived eRNA activates a super-enhancer–driven ALDH1A3/SAT1 axis to promote ferroptosis escape and pancreatic cancer development

doi: 10.1126/sciadv.aea9074

Figure Lengend Snippet: ( A ) RIP was performed in SW1990 cells using a KLF5-specific antibody. Enrichment of HERVH-derived RNAs in the immunoprecipitates was assessed by RT-PCR. n = 3 biological replicates. Data are presented as means ± SD. Statistical significance was evaluated using a two-sided t test. ( B ) CUT&Tag-seq assays were conducted in SW1990 cells following HERVH knockdown using antibodies against KLF5, BRD4, and EP300. Heatmaps display the binding intensities of these proteins within SE regions. ( C and D ) HERVH was knocked down in SW1990 cells, followed by H3K27ac CUT&Tag-seq. ngs.plot was used to visualize changes in H3K27ac signal intensity at (C) SE regions and (D) KLF5 binding sites located within SE regions. ( E ) RT-PCR and Western blot analysis of ALDH1A3, Sp1, and SAT1 expression levels in MIA PaCa-2 cells after treatment with the BRD4 inhibitor JQ1 for 48 hours. n = 3 biological replicates. Data are presented as means ± SD. Statistical significance was determined by a two-sided t test. ( F ) IGV browser visualization of the SE region upstream of the ALDH1A3 gene. ( G and H ) CRISPRi was used to target and repress the SE upstream of ALDH1A3. ALDH1A3 mRNA expression was assessed by RT-PCR (G), and protein levels of ALDH1A3 and SAT1 were evaluated by Western blotting (H). n = 3 biological replicates. Data are shown as means ± SD, and statistical significance was assessed using a two-sided t test. ( I and J ) IGV browser visualization of the ALDH1A3-upstream SE, showing occupancy peaks of BRD4, EP300, and KLF5 within the enhancer region and their changes following HERVH knockdown in SW1990 cells (I). Changes in H3K27ac peak intensity were analyzed in SW1990 and MIA PaCa-2 cells with or without HERVH knockdown to assess SE activity dynamics (J).

Article Snippet: The antibodies used in RIP assays include anti-KLF5 (Proteintech, 21017-1-AP; 5 μg) and anti–immunoglobulin G (IgG) (Cell Signaling Technology Inc., Danvers, MA, USA, #2729; 5 μg).

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Knockdown, Binding Assay, Western Blot, Expressing, Activity Assay