Journal: JCI Insight

Article Title: lncRNA DRAIR is downregulated in diabetic monocytes and modulates the inflammatory phenotype via epigenetic mechanisms

doi: 10.1172/jci.insight.143289

Figure Lengend Snippet: ( A – D ) qPCR analysis of indicated genes in THP1 monocytes (THP1) before and after differentiation into macrophages (TMac) with PMA (20 ng/mL) for 24 hours. * P < 0.05; **** P < 0.0001 as determined by unpaired t test ( n = 3). ( E – H ) Expression of indicated genes in THP1 macrophages treated with IL-4 or IL-13 or a combination of both (20 ng/mL each). * P < 0.05 ( n = 3) as determined by 1-way ANOVA and Dunnett’s multiple comparisons tests. ( I – K ) Gene expression analysis in THP1 cells transiently transfected with control pcDNA3.1 (pCD) or KLF4 expression (pKLF4) plasmids. Gene expression analyses were performed 48 hours after transfection. ** P < 0.01; *** P < 0.001 versus pCD as determined by unpaired t test ( n = 3). ( L ) ChIP-qPCR analysis of ChIP assays with KLF4 antibody with indicated promoter primers (** P < 0.01 versus PPIA promoter, n = 3). ( M ) Schematic of the reporter plasmid (pDRluc) with DRAIR promoter cloned upstream of firefly luciferase reporter gene. KLF4 site (–760) in DRAIR promoter (not to scale). ( N – P ) Luciferase activities with THP1 cells cotransfected with pDRluc and internal control Renilla luciferase. In addition, plasmids pKLF4 and pCDNA3.1 were also cotransfected in P . One day after transfection, cells were treated as indicated for 24 hours. Luciferase activities are reported as fold over controls. In N , Ctrl, control; PA, palmitic acid (200 μM); IL-4, 20 ng/mL; in O , PMA-PMA 20 ng/mL. * P < 0.05, ** P < 0.01 as determined by 1-way ANOVA followed by Dunnett’s multiple-comparison test ( N and P , n = 10–13) and unpaired t test for O ( n = 5).

Article Snippet: Sonicated nuclear lysates were diluted 1:10, and lysates containing equal amounts of DNA were immunoprecipitated using antibodies against H3K9me2 (catalog 4658S), G9a (catalog 68851S), or H3K27me3 (catalog 9733S) obtained from Cell Signaling Technology, or KLF4 (10 μg, catalog AF3640, R&D Systems).

Techniques: Expressing, Gene Expression, Transfection, Control, ChIP-qPCR, Plasmid Preparation, Clone Assay, Luciferase, Comparison

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: KLF4 activates downstream transcription in PC3 and LNCaP cells. (A-C) Regulation of KLF4 on the transcription of miR-7 primary precursors is evaluated by dual-luciferase report assay in PC3 and LNCaP cells. Truncated promoters of pri-miR-7-1 (A), pri-miR-7-2 (B) and pri-miR-7-3 (C) with or without KLF4 binding sites are used to drive luciferase expression in PC3-shKLF4 vs. PC3-con and LNCaP-shKLF4 vs. LNCaP-con cells respectively. **: P<0.01; *: P<0.05.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Luciferase, Binding Assay, Expressing

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: Primers for qRT-PCR

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Sequencing

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: Primary antibodies used in the study

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: Expression of KLF4 and pri-miR-7s is upregulated but that of mature miR-7 is downregulated in clinical patient samples. A. Expression of KLF4 in both patient tumor samples (P1 to P27) and BPH samples (N1 to N9) is measured by qRT-PCR and western blot. B. Expression of KLF4 is upregulated in PCa samples by IHC staining. C. Data from two independent datasets in Oncomine database demonstrates a significant upregulation of KLF4 expression in PCa tissues compared to normal controls. D. Transcription of pri-miR-7s is significantly upregulated in patient tumor samples. E. Expression of mature miR-7 is downregulated in PCa samples. Scale Bar: 50 μm; ***: P<0.001; **: P<0.01.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: Clinical data for 2 benign prostatic hyperplasia (BPH) tissue controls and 2 patient tumor tissues for IHC staining of KLF4 and YAP

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: KLF4 activates the transcription of three miR-7 primary precursors in both PC3 and LNCaP cells. (A) Schematic of predicted KLF4 binding sites on promoter regions of three miR-7 primary precursors. (B, C) Relative enrichment of KLF4 at its binding sites was measured by ChIP assay in PC3 (B) and LNCaP (C) cells. (D) Expression of KLF4 can be inhibited by three independent KLF4 shRNA expressional vectors. The vector expressing shKLF4#1 is selected for construction of KLF4 stable knockdown subclone cell line in LNCaP cells for subsequent experiments due to its best effect on inhibition of KLF4 expression. (E) Knockdown of KLF4 represses expression of both miR-7 primary precursors and mature miR-7 in PC3 and LNCaP cells. **: P<0.01.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Binding Assay, Expressing, shRNA, Plasmid Preparation, Inhibition

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: YAP knockdown restores mature miR-7 production. A. Expression of YAP can be inhibited by three independent siRNAs in both PC3 and LNCaP cells. The best effect is observed by siRNA#2 in both cell lines. B. YAP Knockdown restores p72-Drosha and p72-DGCR8 interaction. C. YAP knockdown has no effect on expression of pri-miR-7s. D. Expression of mature miR-7 is upregulated after YAP knockdown. E. Expression of KLF4 is downregulated after YAP knockdown. F. Expression of Dicer has no difference in BPH-1, PC3 and LNCaP cells. **: P<0.01.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: Overexpression of mature miR-7 mimics inhibits cell proliferation in LNCaP cells. A. Expression of mature miR-7 is upregulated and expression of KLF4 is downregulated in LNCaP cells after transfected with miR-7 mimics. B. Proliferation of LNCaP cells is detected by CCK8 assay after overexpression of mature miR-7 mimics or knockdown of KLF4. C. Proliferation of LNCaP cells is detected by 2-D plate colony formation assay after overexpression of mature miR-7 mimics or knockdown of KLF4. D. Overexpression of mature miR-7 mimics or knockdown of KLF4 inhibits 3-D sphere formation of LNCaP cells. Scale bar: 100 μm. **: P<0.01.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Over Expression, Expressing, Transfection, CCK-8 Assay, Colony Assay

Journal: American Journal of Cancer Research

Article Title: Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing

doi:

Figure Lengend Snippet: The KLF4-miR-7 auto-regulatory feedback loop is unbalanced in PCa cells. Enhanced nuclear translocation of oncogenic YAP sequestrates p72 to dissociate the p72/Drosha/DGCR8 microprocessor complex, leading to a decreased production of mature miR-7 and an upregulation of its target KLF4. Up or down white arrow: upregulation or downregulation of gene expression.

Article Snippet: KLF4 shRNA (TG316853) expression vector and control vector (TR30013) were purchased from Origene (Rockville, MD, USA).

Techniques: Translocation 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: 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: 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