Journal: PLoS ONE

Article Title: Cancer stem cell subpopulations in primary colon adenocarcinoma

doi: 10.1371/journal.pone.0221963

Figure Lengend Snippet: Representative 3,3-diaminobenzidine immunohistochemical-stained images showing protein expression of induced-pluripotent stem cell markers OCT4 (A-C, brown), SOX2 (D-F, brown), NANOG (G-I, brown), KLF4 (J-L, brown) and c-MYC (M-O, brown) in normal colon (A,D,G,J,M), low-grade (B,F,H,K,N) and high-grade (C,F,I,L,O) colon adenocarcinoma tissue samples. Nuclei were counter-stained with hematoxylin (A-O, blue). Original magnification: 400x.

Article Snippet: Staining was carried out on the Leica BOND TM RX Auto-stainer using primary antibodies for OCT4 (1:30; cat#MRQ-10, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat#ab97959, Abcam, Cambridge, MA, USA), NANOG (1:200; cat#EP225, Cell Marque), KLF4 (1:200; cat#NBP2-24749SS, Novus Biologicals LLC, Littleton, CO, USA) and c-MYC (1:1000; cat#ab32, Abcam).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: PLoS ONE

Article Title: Cancer stem cell subpopulations in primary colon adenocarcinoma

doi: 10.1371/journal.pone.0221963

Figure Lengend Snippet: Percentage of cell population stained positively for induced-pluripotent stem cell markers OCT4, SOX2, NANOG, KLF4 and c-MYC by 3,3-diaminobenzidine immunohistochemical staining, for the epithelium (A) and the stroma (B). Normal colon samples from patients with low-grade colon adenocarcinoma (LGCA; pale blue, n = 9) are displayed separately to normal colon samples from patients with high-grade colon adenocarcinoma (HGCA; dark blue, n = 8). LGCA samples are shown in pale yellow (n = 10), and HGCA samples are shown in dark yellow (n = 8). Statistical significance with a p -value between 0.05 and 0.01 is shown by *, and that for <0.01 is represented by **. Error bars show standard error.

Article Snippet: Staining was carried out on the Leica BOND TM RX Auto-stainer using primary antibodies for OCT4 (1:30; cat#MRQ-10, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat#ab97959, Abcam, Cambridge, MA, USA), NANOG (1:200; cat#EP225, Cell Marque), KLF4 (1:200; cat#NBP2-24749SS, Novus Biologicals LLC, Littleton, CO, USA) and c-MYC (1:1000; cat#ab32, Abcam).

Techniques: Staining, Immunohistochemical staining

Journal: PLoS ONE

Article Title: Cancer stem cell subpopulations in primary colon adenocarcinoma

doi: 10.1371/journal.pone.0221963

Figure Lengend Snippet: Representative immunofluorescence immunohistochemical-stained images showing protein expression of induced-pluripotent stem cell markers OCT4 (A-J, green), KLF4 (A-C, red), NANOG (D-G, H-J, red), SOX2 (H-J, red), and c-MYC (H-J, green) in normal colon (A,D,E,H), low-grade (B,E,F,I) and high-grade (C,F,G,J) colon adenocarcinoma tissue samples. Cell nuclei were counter-stained with 4’, 6’-diamidino-2-phenylindole (A-L, blue). Original magnification: 400x.

Article Snippet: Staining was carried out on the Leica BOND TM RX Auto-stainer using primary antibodies for OCT4 (1:30; cat#MRQ-10, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat#ab97959, Abcam, Cambridge, MA, USA), NANOG (1:200; cat#EP225, Cell Marque), KLF4 (1:200; cat#NBP2-24749SS, Novus Biologicals LLC, Littleton, CO, USA) and c-MYC (1:1000; cat#ab32, Abcam).

Techniques: Immunofluorescence, Immunohistochemical staining, Staining, Expressing

Journal: PLoS ONE

Article Title: Cancer stem cell subpopulations in primary colon adenocarcinoma

doi: 10.1371/journal.pone.0221963

Figure Lengend Snippet: mRNA expression of induced-pluripotent stem cell markers OCT4 (A), SOX2 (B), NANOG (C), KLF4 (D) and c-MYC (E) detected by RT-qPCR. Data displayed as the fold-change of gene expression in tumor samples relative to their patient-matched normal colon sample (Y-axis). A cohort of six LGCA tissue samples are shown in blue, and a cohort of six HGCA tissue samples are shown in yellow (X-axis).

Article Snippet: Staining was carried out on the Leica BOND TM RX Auto-stainer using primary antibodies for OCT4 (1:30; cat#MRQ-10, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat#ab97959, Abcam, Cambridge, MA, USA), NANOG (1:200; cat#EP225, Cell Marque), KLF4 (1:200; cat#NBP2-24749SS, Novus Biologicals LLC, Littleton, CO, USA) and c-MYC (1:1000; cat#ab32, Abcam).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: PLoS ONE

Article Title: Cancer stem cell subpopulations in primary colon adenocarcinoma

doi: 10.1371/journal.pone.0221963

Figure Lengend Snippet: Representative images of in-situ hybridization, showing mRNA expression of iPSC genes OCT4 (A-C, brown), SOX2 (D-F, brown), NANOG (G-I, brown), KLF4 (J-L, brown) and c-MYC (M-O, brown) in the epithelial cells ( arrows ) and stromal cells ( arrowheads ) in normal colon (A,D,G,J,M), low-grade (B,E,H,K,N) and high-grade (C,F,I,L,O) colon adenocarcinoma tissue samples. Nuclei were counter-stained with hematoxylin (blue). Original magnification: 1000x.

Article Snippet: Staining was carried out on the Leica BOND TM RX Auto-stainer using primary antibodies for OCT4 (1:30; cat#MRQ-10, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat#ab97959, Abcam, Cambridge, MA, USA), NANOG (1:200; cat#EP225, Cell Marque), KLF4 (1:200; cat#NBP2-24749SS, Novus Biologicals LLC, Littleton, CO, USA) and c-MYC (1:1000; cat#ab32, Abcam).

Techniques: In Situ Hybridization, Expressing, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Pharmacological inhibition of FOXO1 promotes lymphatic valve growth in a congenital lymphedema mouse model

doi: 10.3389/fcell.2022.1024628

Figure Lengend Snippet: Western blot antibodies.

Article Snippet: KLF4 , NBP217070 (Novus Biologicals) , 1:333.

Techniques: Western Blot, Diagnostic Assay

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: The Galectin-3 promoter is bound and transactivated by KLF4. (A) Heatmap analysis of transcription factor activity predicted by the SCENIC package for proximal tubular cells across the indicated groups. (B) The potential promoter sequences of Galectin-3 bound by the transcription factor KLF4, as predicted by the JASPAR database. (C) Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assays showing PCR amplification of Galectin-3 chromatin corresponding to the region of the promoter (site1: nucleotides -1991 to -2000 and site2: -1443 to -1452 presented in (B)) immunoprecipitated with anti-KLF4 or with control IgG antibody from cisplatin-challenged kidneys. (D) Western blot and graphic presentation showing changes in KLF4 expression in HK2 cells treated with cisplatin (25 μg/ml) or H 2 O 2 (500 mM) at different time points as indicated. (E) Western blot for KLF4 and Galectin-3 protein in HK2 cells transfected with scramble or KLF4 siRNA and followed by cisplatin (25 μg/ml) for 12 hours. One of the three independent experiments with identical results was shown. (F) Representative immunofluorescence staining images for Galectin-3 in HK2 cells transfected with scramble or KLF4 siRNA. Scale bar = 25 μm. (G) KLF4 protein expression in HK2 cells transfected with KLF4-OE plasmid. One of the three independent experiments with identical results was shown. (H) Galectin-3 protein levels in HK2 cells transfected with empty vector control or KLF4-OE plasmid followed by cisplatin treatment. (I) Semiquantitative analysis of Galectin-3 protein from (H) (n = 3). (J) Schematic illustration of Galectin-3 promoter reporter constructs containing the wild-type KLF4 binding sequences (BS WT) and the corresponding mutant sequences (BS Mut) used in luciferase assays. (K) Relative activation of WT and mutant Galectin-3 promoter by KLF4 in 293T cells. The luciferase activity of each group was normalized to that co-transfected with pECMV-NC and pgl4 plasmid (n = 4). Data are presented as means ± SEM. * p ˂0.05 or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Control, Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Plasmid Preparation, Construct, Binding Assay, Mutagenesis, Luciferase, Activation Assay

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: Blocking the KLF4/Galectin-3 signaling cascade attenuates kidney tubular cell death. (A) Western blot for cleaved PARP and cleaved caspase 3 protein in HK2 cells transfected with scramble or KLF4 siRNA followed by cisplatin exposure. (B) Semiquantitative analysis for cleaved PARP and cleaved caspase 3 protein from (A) (n = 3). (C) Representative flow cytometry plots analyzing KLF4 and scramble siRNA- treated HK2 cells, which were then treated with either vehicle or cisplatin. (D) Summary data quantifying apoptosis among different groups in (C) (n = 3). (E-F) Propidium Iodide (PI) staining assay (E) and quantitative analysis (F) of PI in HK2 cells among groups indicated. Scale bar = 50 μm. (G) Western blot for cleaved PARP and cleaved caspase 3 in HK2 cells with pECMV-KLF4 and/ or Galectin-3 siRNA followed by cisplatin treatment. (H) Semiquantitative analysis for cleaved PARP and cleaved caspase 3 protein levels from (G) (n = 3). (I) KLF4 and Galectin-3 protein expression in HK2 cells pretreated with Kenpaullone and followed by cisplatin (25 μg/ml) for 12 hrs. One of the three independent experiments with identical results was shown. (J) Western blot showing cleaved PARP and cleaved caspase 3 protein levels in HK2 cells pretreated with different concentrations of Kenpaullone followed by cisplatin exposure. (K) Representative flow cytometry plots and quantitative analyses of Kenpaullone- and vehicle-treated HK2 cells, as indicated by the groups (n = 3). (L) PI staining assay and quantitative analysis of PI in HK2 cells among groups indicated (n=3). Scale bar = 50 μm. (M) Western blots showing KLF4 and Galectin-3 protein expression in HK2 cells for the indicated group. One of the three independent experiments with identical results was shown. (N) Western blot analysis showing the levels of cleaved PARP and cleaved caspase 3 proteins in HK2 cells pretreated with various concentrations of APTO-253 before exposure to cisplatin. (O-P) PI staining assay (O) and quantitative analysis (P) of PI in HK2 cells among indicated groups (n=3). Scale bar = 50 μm. Ken, Kenpaullone; PI, Propidium Iodide. Data are presented as means ± SEM. * p ˂0.05, ** p ˂0.01, or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Blocking Assay, Western Blot, Transfection, Flow Cytometry, Staining, Expressing

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: KLF4 protein is induced in tubular cells from patients and mouse models with acute kidney injury, and deletion of KLF4 in proximal tubular cells attenuates cisplatin-induced AKI. (A) Representative immunohistochemical staining images showing the expression of KLF4 in kidney tubular cells from patients with acute kidney injury (AKI). Red arrows indicating the KLF4 positive tubular cells. Scale bar = 50 μm. (B) Representative staining images showing colocalization of KLF4 and Galectin-3 proteins in kidney sections from patients with AKI. White arrow heads indicating double positive tubular cells. Scale bar = 50 μm. (C-D) Western blot assay (C) and semiquantitative analysis (D) showing the abundance of KLF4 protein in the mouse kidneys after cisplatin exposure at day 2 and 3 (n = 3). (E) Linear regression analysis of KLF4 and Galectin-3 expression levels in the kidneys of cisplatin mouse model. (F-G) Western blot assay (F) and semiquantitative analysis (G) showing the expression of KLF4 in the kidneys after IRI at day 1 and 3 (n = 3). (H) Linear regression analysis of KLF4 and Galectin-3 expression levels in the kidneys of IRI mouse model. (I) Strategy for generating mice with kidney proximal tubular-specific deletion of KLF4. (J) Genotyping the mice by PCR analysis of genomic DNA. (K) Representative immunofluorescence staining for KLF4 protein in WT and PKO kidney sections after cisplatin treatment. Scale bar = 50 μm. (L-O) KLF4 and Galectin-3 mRNA (L-M) and protein (N-O) expression levels in kidneys from WT and PKO mice following cisplatin exposure (n = 5). (P) Kidney histology from the groups as shown by PAS staining and kidney pathology scores (n = 5). Scale bar =100 μm. (Q) Serum creatinine and BUN among groups as indicated (n = 5). AKI, acute kidney injury; IRI, ischemia-reperfusion injury. Data are presented as means ± SEM. * p ˂0.05, ** p ˂0.01, or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: Tubular KLF4 deficiency attenuates kidney injury, apoptosis and inflammatory response. (A) Principal component analysis of global transcriptomics from WT and PKO kidneys following cisplatin challenge. (B) Heatmap of significant gene expression from WT and PKO kidneys with cisplatin exposure. (C) Renal mRNA expression levels of KIM-1, NGAL and Hnf4a in cisplatin-exposed kidneys from WT and PKO mice (n = 5). (D) Western blots for KIM-1, NGAL and cleaved caspase 3 in kidneys from WT and MKO mice after cisplatin injection at day 3. (E) Semiquantitative determination of protein abundance in (D) (n = 5). (F) Representative kidneys stained with KIM-1 and NGAL protein. Scale bar = 50 μm. (G) Representative images and quantification of TUNEL staining in kidney sections from WT and PKO mice with cisplatin nephropathy (n = 5). Scale bar = 50 μm. (H) Representative immunofluorescence staining for F4/80 and Ly6G in cisplatin-exposed kidneys from different groups as indicated. Scale bar = 50 μm. (I) Quantitative analysis for F4/80-positive macrophages and Ly6G-positive neutrophils in cisplatin-exposed kidneys among groups as indicated (n = 5). (J) The IL6, TNFa, and MCP-1 mRNA expression levels in WT and PKO kidneys following cisplatin treatment (n = 5). Data are presented as means ± SEM. * p ˂0.05, ** p ˂0.01, or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Gene Expression, Expressing, Western Blot, Injection, Quantitative Proteomics, Staining, TUNEL Assay, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: KLF4 deletion in proximal tubular cells ameliorates IRI-induced kidney injury and inflammatory response. (A) Representative images for PAS staining in kidneys among groups as indicated. Scale bar = 100 μm. (B) Kidney pathology scores in (A) (n = 5). (C) Serum creatinine and BUN levels in WT and PKO mice following IRI (n = 5). (D) Renal mRNA levels for KLF4 and Galectin-3 in IRI model (n = 5). (E) Western blot assay and semiquantitative analysis for Galectin-3 protein in IRI kidneys from different groups as indicated (n = 5). (F) Representative immunohistochemical staining for Galectin-3 protein in IRI kidneys among groups as indicated. Scale bar = 50 μm. (G-H) Western blot assay (G) and semiquantitative analysis (H) for KIM-1, NGAL and cleaved caspase 3 protein in IRI kidneys (n = 5). (I) Representative immunohistochemical staining for KIM-1 and NGAL protein in IRI kidneys among groups as indicated. Scale bar = 50 μm. (J-K) Representative images (J) and quantitative analysis (K) of TUNEL staining in the indicated groups (n = 5). Scale bar = 50 μm. (L-M) Representative immunochemical staining (L) and quantitative analysis of F4/80 and Ly6G (M) in IRI-induced kidneys from the indicated groups (n = 5). Scale bar = 50 μm. (N) Renal mRNA expression levels for IL6, TNFa and MCP-1 in IRI kidneys among groups as indicated (n = 5). Data are presented as means ± SEM. * p ˂0.05, ** p ˂0.01, or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Staining, Western Blot, Immunohistochemical staining, TUNEL Assay, Expressing

Journal: International Journal of Biological Sciences

Article Title: The KLF4/Galectin-3 cascade is a key determinant of tubular cell death and acute kidney injury

doi: 10.7150/ijbs.110790

Figure Lengend Snippet: Inhibition of KLF4 signaling with Kenpaullone attenuates cisplatin-induced acute kidney injury. (A-B) Western blot assay (A) and semiquantitative analysis (B) for KLF4 and Galectin-3 protein in cisplatin-exposed kidneys among groups as indicated (n = 6). (C) Representative images for PAS staining in cisplatin-exposed kidneys and kidney pathology scores (n = 6). Scale bar = 100 μm. (D) Serum creatinine and BUN levels in groups as indicated (n = 6). (E-F) Western blot assay (E) and semiquantitative analysis (F) for KIM-1, NGAL and cleaved caspase 3 protein in cisplatin-exposed kidneys among groups as indicated (n = 6). (G) Representative images for KIM-1 and NGAL staining in cisplatin-exposed kidneys from vehicle and Kenpaullone-treated mice. Scale bar = 50 μm. (H-I) TUNEL staining (H) and quantification analysis (I) of kidney sections from vehicle and Kenpaullone-treated mice following cisplatin exposure (n = 6). Scale bar = 50 μm. (J-K) Representative immunofluorescence staining (J) and quantitative analysis (K) of F4/80 and Ly6G in cisplatin-treated kidneys from the indicated groups (n = 5). Scale bar = 50 μm. (L) IL-6, TNFa, and MCP-1 mRNA abundance in vehicle and Ken-treated kidneys following cisplatin exposure (n = 6). Data are presented as means ± SEM. * p ˂0.05, ** p ˂0.01, or *** p ˂0.001.

Article Snippet: Mice with a floxed KLF4 allele were ordered from Cyagen (cat: S-CKO-03285, Guangzhou, China, C57BL/6J background).

Techniques: Inhibition, Western Blot, Staining, TUNEL Assay, Immunofluorescence

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner

doi: 10.1016/j.mcpro.2021.100064

Figure Lengend Snippet: KLF4 depletion does not affect AR expression but increases its transcriptional activity. A , control, siAR, siGRHL2, siMAML1, siKLF4, siRBPJ, and siTBL1X transfected LAPC4 cells were infected with a luciferase reporter gene coupled with a PSA promoter and treated with a vehicle or 10 nM DHT. Luciferase activity was normalized to the total protein amount for each sample and to the vehicle-treated condition to obtain relative luciferase intensities for each sample (∗ p ≤ 0.05; ∗∗∗ p ≤ 0.001). B , western blot analysis of 3xFLAG-KLF4 following GFP affinity purification in HEK293 T cells cotransfected with GFP/GFP-AR and 3xFLAG-KLF4. C–D , Control, siAR, or siKLF4 transfected LAPC4 cells were infected with a luciferase reporter gene coupled with a PSA promoter and treated with a vehicle or 10 nM DHT. Normalization was performed as in ( A ). Endogenous AR- or KLF4-depleted cells were analyzed by western blot ( C ) to confirm protein depletion. E and F , Control, siAR, or siKLF4 transfected LAPC4 cells were stimulated with a vehicle or 10 nM DHT, and RT-qPCR was performed to assess AR ( E ) and KLF4 ( F ) mRNA levels. Data was normalized to actin mRNA levels, and then to the control condition to obtain relative values. G and H , Control, siAR, or siKLF4 transfected LAPC4 cells were infected with a luciferase reporter gene coupled with a PSA promoter, transfected with Flag-BirA or Flag-KLF4, and treated with a vehicle or 10 nM DHT. Endogenous AR- or KLF4-depleted LAPC4 cells were analyzed by western blot ( G ) to determine protein levels. Luciferase activity was normalized to the total protein amount for each sample and to the vehicle-treated condition to obtain relative luciferase intensities for each sample ( H ). I and J , control, siAR, or siKLF4 transfected VCaP cells were infected with a luciferase reporter gene coupled with a PSA promoter and treated with a vehicle or 10 nM DHT. Endogenous AR- or KLF4-depleted cells were analyzed by western blot ( I ) to confirm protein depletion. Luciferase activity was normalized to the total protein amount for each sample and to the vehicle-treated condition to obtain relative luciferase intensities for each sample ( J ). D, E, F, H, J , Mean values and standard deviation from three independent experiments are presented (∗ p ≤ 0.05; ∗∗∗∗ p ≤ 0.0001). Blots in B , C , and G are representative of three independent experiments, blots in I are representative of two independent experiments.

Article Snippet: Sonicated DNA fragments were immunoprecipitated with antibodies directed against KLF4 (R&D Systems, AF3640) and AR (Sigma, EMB Millipore, #06–680).

Techniques: Expressing, Activity Assay, Control, Transfection, Infection, Luciferase, Western Blot, Affinity Purification, Quantitative RT-PCR, Standard Deviation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner

doi: 10.1016/j.mcpro.2021.100064

Figure Lengend Snippet: A large fraction of KLF4 colocalizes with AR in the genome and acts as a repressor of the AR target gene KLK3 ( PSA ) in LAPC4 cells. A–C , control, siAR, or siKLF4 transfected LAPC4 cells were stimulated with a vehicle or 10 nM DHT, and RT-qPCR was performed to assess PSA mRNA levels ( A ). AR ( B ) and KLF4 ( C ) depletion efficiency was also determined by RT-qPCR. Data was normalized to actin mRNA levels, and then to the control condition to obtain relative values. Mean values and standard deviation from three independent experiments are presented (∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001). D , heat map showing the co-occupancy of KLF4 and AR across the genome of LAPC4 cells. Top : Density heat maps representing KLF4 and AR ChIP-seq intensities in the absence (control) or presence of DHT at three groups of genomics regions (occupied by AR and KLF4 (n = 35,709), occupied by KLF4 only (n = 24,190), and occupied by AR only (n = 49,877)). Regions were ranked according to the total read density in the KLF4 control condition. Color scales indicate bins per million mapped reads (BPM). Bottom: Average read density plots for the same groups of regions. A region of 5kb centered on the occupied region is displayed. E , ChIP-Seq occupancy profiles of KLF4 and AR in control and DHT-stimulated cells. Genome tracks show colocalization of KLF4 ( blue ) and AR ( yellow ) ChIP-Seq peaks in the neighborhood of KLK3 ( PSA ), KLK2 , SGK1 , and IDH1 genes that are known to be regulated by AR. ChIP-Seq profiles are displayed in BPM. Gene depictions are presented below the gene tracks.

Article Snippet: Sonicated DNA fragments were immunoprecipitated with antibodies directed against KLF4 (R&D Systems, AF3640) and AR (Sigma, EMB Millipore, #06–680).

Techniques: Control, Transfection, Quantitative RT-PCR, Standard Deviation, ChIP-sequencing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner

doi: 10.1016/j.mcpro.2021.100064

Figure Lengend Snippet:

Article Snippet: Sonicated DNA fragments were immunoprecipitated with antibodies directed against KLF4 (R&D Systems, AF3640) and AR (Sigma, EMB Millipore, #06–680).

Techniques: Sequencing

Journal: Cell Death & Disease

Article Title: ACTL6A depletion induces KLF4-mediated anti-tumorigenic effects in colorectal cancer

doi: 10.1038/s41419-025-07946-w

Figure Lengend Snippet: A Co-immunoprecipitation (co-IP) experiments using antibodies against SMARCB1, SMARCC1, and INO80 in whole-cell extracts from HCT116 ells transduced with sgCtrl or sgACTL6A. Western blotting results of ACTL6A (SWI/SNF and INO80 complex subunit), SMARCA4, SS18, SMARCC1, SMARCB1 (SWI/SNF complex subunits), INO80, and YY1 (INO80 complex subunits). Arrows indicate SS18 and SS18/IgG. IgG bands are marked by orange asterisks (*). The black asterisk (*) indicates the INO80 band. B Heatmaps of GAIN and LOSS regions following ACTL6A depletion, showing ATAC-seq, SMARCB1, INO80, KLF4, and RNA Pol II-S2P ChIP-seq signals. C Correlation plot indicating the change in accessibility and SMARCB1 (top) and INO80 (bottom) enrichment. Red and blue indicate the GAIN and LOSS regions, respectively. Pearson’s method was used to calculate correlation coefficients. D Pie chart illustrating the distribution of SMARCB1 and INO80 enrichment in the GAIN regions. E IGV tracks of SMARCB1, INO80, RNA PII-S2P enrichment (ChIP-seq) and chromatin accessibility (ATAC-seq) at the SERPINB5 and S100A10 loci in HCT116 cells transduced with sgCtrl or sgACTL6A.

Article Snippet: The LentiCRISPRv2-blast vector (#98293; Addgene) was used to deplete ACTL6A in cells expressing CRISPR-resistant ACTL6A. pBpuro-HA KLF4 FL (#34589; Addgene) was used for KLF4 overexpression.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Transduction, Western Blot, ChIP-sequencing

Journal: Cell Death & Disease

Article Title: ACTL6A depletion induces KLF4-mediated anti-tumorigenic effects in colorectal cancer

doi: 10.1038/s41419-025-07946-w

Figure Lengend Snippet: A Bar plot illustrating the results of motif analysis. Red and blue indicate the GAIN and LOSS, respectively. Asterisks (*) indicate the DEGs. B TCGA-COAD datasets were used to compare the differences in KLF4 expression between matched normal and tumor tissues. Expression values were determined using VST with the DESeq2 package. C Venn diagram illustrating the overlap between the KLF4 target genes based on the ChIP Enrichment Analysis database and the upregulated genes induced by ACTL6A depletion. D The overlapping genes shown in ( C ) were used for functional enrichment analysis based on the Hallmark gene set from the Molecular Signatures Database. E Heatmaps of GAIN and LOSS regions following ACTL6A depletion, showing KLF4 ChIP-seq signals. F Histogram illustrating KLF4 tags per base pair per peak from −3000 bp upstream to +3000 bp downstream of the identified GAIN regions upon ACTL6A depletion in HCT116 cells. G Correlation plot illustrating changes in accessibility and KLF4 enrichment. Red and blue indicate the GAIN and LOSS, respectively. Pearson’s analysis was used to calculate the correlation coefficients. H Co-IP experiments using the KLF4 antibody on whole-cell extracts from HCT116, LS174T, and LoVo cells. Western blotting results of KLF4 and ACTL6A. IgG bands are marked by black asterisks (*). I Co-IP experiments using KLF4 antibody in whole-cell extracts from HCT116 cells transduced with sgCtrl or sgACTL6A. Western blotting results of KLF4, ACTL6A (SWI/SNF and INO80 complex subunit), SMARCA4, SMARCB1, SMARCC1 (SWI/SNF complex subunits), INO80 (INO80 complex subunit). IgG bands are marked by black asterisks (*).

Article Snippet: The LentiCRISPRv2-blast vector (#98293; Addgene) was used to deplete ACTL6A in cells expressing CRISPR-resistant ACTL6A. pBpuro-HA KLF4 FL (#34589; Addgene) was used for KLF4 overexpression.

Techniques: Expressing, Functional Assay, ChIP-sequencing, Co-Immunoprecipitation Assay, Western Blot, Transduction

Journal: Cell Death & Disease

Article Title: ACTL6A depletion induces KLF4-mediated anti-tumorigenic effects in colorectal cancer

doi: 10.1038/s41419-025-07946-w

Figure Lengend Snippet: A Western blotting of the indicated antibodies in HCT116, LS174T, and LoVo cells transduced with an empty or HA-tagged KLF4-expressing vector. β-Actin was used as the loading control. B Relative cell viability of HCT116, LS174T, and LoVo cells transduced with an empty or HA-tagged KLF4-expressing vector. Cell viability was determined using an ATP assay. ATP levels (y-axis) were measured using CellTiter-Glo. n = 3 biological replicates for HCT116 and LoVo, n = 4 biological replicates for LS174T. ** p < 0.01, **** p < 0.0001. C Normalized mRNA expression of P53 pathway-related genes in HCT116, LS174T, and LoVo cells transduced with an empty or HA-tagged KLF4-expressing vector. n = 3 biological replicates for HCT116 and LS174T, n = 4 biological replicates for LoVo. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. D Schematic of luciferase reporter constructs containing wild-type (WT) or mutant (Mut) KLF4 binding motifs upstream of KLF4 , SERPINB5 , or DGKA promoters cloned into pGL3-basic vector. E Dual luciferase reporter assays were performed to assess KLF4 , SERPINB5 , or DGKA promoter activity in HCT116 cells transduced with sgCtrl or sgACTL6A. Firefly luciferase activity was normalized to Renilla luciferase to control for transfection efficiency. n = 3 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001. F Western blotting using the indicated antibodies in HCT116 and LS174T cells transduced with indicated sgRNAs. β-Actin was used as the loading control. G Annexin V/propidium iodide assay for apoptosis in HCT116 and LS174T cells transduced with indicated sgRNAs. Statistical analysis (left) and representative flow cytometry plots (right) of sgACTL6A or sgKLF4+sgACTL6A (DKO) (right). n = 3 biological replicates. ** p < 0.01. H Relative cell viability of HCT116 and LS174T transduced with indicated sgRNAs. Cell viability was determined using an ATP assay. ATP levels (y-axis) were measured using CellTiter-Glo. n = 4 biological replicates. * p < 0.05, ** p < 0.01. I Normalized mRNA expression of the indicated genes in HCT116 and LS174T cells transduced with indicated sgRNAs, as determined by RT-qPCR. n = 3 biological replicates for HCT116, n = 4 biological replicates for LS174T. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The LentiCRISPRv2-blast vector (#98293; Addgene) was used to deplete ACTL6A in cells expressing CRISPR-resistant ACTL6A. pBpuro-HA KLF4 FL (#34589; Addgene) was used for KLF4 overexpression.

Techniques: Western Blot, Transduction, Expressing, Plasmid Preparation, Control, ATP Assay, Luciferase, Construct, Mutagenesis, Binding Assay, Clone Assay, Activity Assay, Transfection, Flow Cytometry, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: ACTL6A depletion induces KLF4-mediated anti-tumorigenic effects in colorectal cancer

doi: 10.1038/s41419-025-07946-w

Figure Lengend Snippet: A Pie chart illustrating the distribution of SMARCB1, INO80, and KLF4 binding sites based on ChIP-seq data. B Heatmaps revealing alterations in normalized tag values for P53 pathway-related GAIN regions upon ACTL6A depletion in HCT116 cells. The first three columns represent the ATAC-seq replicates (n = 3, green), followed by two columns for the SMARCB1 ChIP-seq replicates (n = 2, light purple), two colums for INO80 ChIP-seq replicates (n = 2, light coral), and two columns for KLF4 ChIP-seq replicates (n = 2, aqua). Changes in the normalized tag values were calculated as log2(sgACTL6A + 1) − log2(sgCtrl + 1). C Histogram revealing the ATAC-seq tags per base pair per peak from −3000 bp upstream to +3000 bp downstream of the identified GAIN regions in the HCT116 cells transduced with sgACTL6A or sgKLF4+sgACTL6A (DKO). D Heatmaps showing the changes in normalized tag values for the P53 pathway-related GAIN regions upon ACTL6A depletion in HCT116 cells. The first three columns represent the ATAC-seq replicates for log2(sgACTL6A + 1) − log2(sgCtrl + 1), whereas the next two columns show the ATAC-seq replicates for log2(DKO + 1) − log2(sgACTL6A + 1). E IGV tracks of SMARCB1, INO80, KLF4, and RNA PII-S2P enrichment (ChIP-seq) and chromatin accessibility (ATAC-seq) at the CTSD and KLF4 loci in HCT116 cells transduced with indicated sgRNAs. F ChIP-qPCR for KLF4, SMARCB1 and INO80 at the GAIN region upstream of the KLF4 (KLF4-GAIN) in HCT116 cells transduced with sgACTL6A or sgKLF4+sgACTL6A (DKO). n = 3 biological replicates. ** p < 0.01. G Sanger sequencing showing a 869 bp deletion in KLF4-GAIN region, two sgRNA target sequences (black and gray text), and a protospacer adjacent motif (PAM) sequence (lowercase). H Western blotting results of ACTL6A in HCT116 cells transduced with indicated sgRNAs. β-Actin was used as the loading control. I Normalized mRNA expression of KLF4 in HCT116 cells transduced with indicated sgRNAs. n = 3 biological replicates. * p < 0.05, ** p < 0.01, **** p < 0.0001. J ChIP-qPCR for KLF4, SMARCB1 and INO80 at the GAIN region upstream of the KLF4 (KLF4-GAIN) in HCT116 cells transduced with indicated sgRNAs. n = 3 biological replicates. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The LentiCRISPRv2-blast vector (#98293; Addgene) was used to deplete ACTL6A in cells expressing CRISPR-resistant ACTL6A. pBpuro-HA KLF4 FL (#34589; Addgene) was used for KLF4 overexpression.

Techniques: Binding Assay, ChIP-sequencing, Transduction, ChIP-qPCR, Sequencing, Western Blot, Control, Expressing