Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: Patients with transplant-associated thrombotic microangiopathy exhibit abnormal complement activation and endothelial injury. (A) Plasma levels of C3b (left panel), sC5b-9 (center panel), and intercellular cell adhesion molecule-1 (right panel) in patients with transplant-associated thrombotic microangiopathy (TA-TMA) and controls. N=20 per group. (B) In the left, center, and right panels: plasma levels of vascular cell adhesion molecule-1, plasminogen activator inhibitor-1, and suppression of tumorigenicity 2, respectively, in TA-TMA patients and controls. N=20 per group. (C) Plasma levels of Krüppel-like factor 4 (KLF4) in TA-TMA patients versus controls. N=20 per group. (D) MA plot of complement/endothelium-associated differentially expressed genes (red: upregulated; blue: downregulated; gray: not significantly different; dashed horizontal lines: P -value thresholds) in human umbilical vein endothelial cells (HUVEC) and circulating endothelial cells (CEC) from RNA-sequencing analysis. N=3 per group. (E) Western blot analysis of KLF4 protein levels in HUVEC and CEC. The bar graph shows the relative intensity of KLF4. The results are representative of three experiments. N=4 per group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, NS: not statistically significant. C3b: complement 3b fragment; sC5b-9: soluble complement factors; ICAM-1: intercellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; PAI-1: plasminogen activator inhibitor-1; ST2: suppression of tumorigenicity 2.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Activation Assay, Clinical Proteomics, RNA Sequencing, Western Blot

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: KLF4 overexpression reduces complement deposition and endothelial injury under challenge with transplant-associated thrombotic microangiopathy plasma. (A, B) Flow cytometric detection of C3 (A) and C5b-9 (B) in KLF4-overexpressing human umbilical vein endothelial cells (HUVEC) (OEKLF4) and control lentiviral-transduced cells (PCDH) incubated with control (Ctrl) or transplant-associated thrombotic microangiopathy (TMA) plasma. The bar graphs show mean fluorescence intensities. (C) Western blot of complement proteins (C3, C5, C4, CFB, MBL2, C1QA) in these cells. The bar graphs compare the relative intensities of C3, C5 and C4. (D) Relative decrease (ΔTEER) of transendothelial electrical resistance in the indicated groups. (E) Fluorescein isothiocyanate-dextran permeability assay reflecting endothelial barrier integrity. (F) Flow cytometric analysis of apoptosis in PCDH and OEKLF4 cells treated with control or TMA plasma. The bar graph shows the quantification of apoptotic cells. The results are representative of three experiments. * P <0.05, *** P <0.001, **** P <0.0001.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Over Expression, Clinical Proteomics, Control, Incubation, Fluorescence, Western Blot, FITC-Dextran Permeability Assay

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: KLF4 transcriptionally activates CD46 gene expression in human umbilical vein endothelial cells. (A) Integrative Genomics Viewer tracks of CUT&Tag showing enriched KLF4 in the promoter of CD46 . (B) Quantitative real-time polymerase chain reaction (qPCR) of genomic DNA isolated by CUT&Tag, confirming KLF4 enrichment at the CD46 promoter. (C) Interaction between CD46 promoter and KLF4 by dual luciferase reporter assays. The results are representative of three experiments. (D) CD46 mRNA levels measured by qPCR in KLF4-overexpressing (OEKLF4) and control lentiviral-transduced cells (PCDH) exposed to transplant-associated thrombotic microangiopathy (TMA) plasma. The results are representative of three experiments. (E) Jaspar-based prediction of three KLF4-binding sites in the CD46 promoter identifies transcriptional regulatory elements. (F) Basenji in silico knockout identifies sites 2 and 3 as critical regulators of CD46 transcriptional activity. ** P <0.01, *** P <0.001, **** P <0.0001. Refseq: reference sequence; IgG: immunoglobulin G; TF: transcription factors.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Isolation, Luciferase, Control, Clinical Proteomics, Binding Assay, In Silico, Knock-Out, Activity Assay, Sequencing

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: KLF4-mediated protection is abrogated by CD46 knockdown. (A, B) Flow cytometric measurement of C3 (A) and C5b-9 (B) deposition in CD46 knockdown human umbilical vein endothelial cells (ShCD46) or control cells (PLKO.1) treated with control plasma, transplant-associated thrombotic microangiopathy (TMA) plasma, or TMA plasma plus APTO253. The bar graphs show mean fluorescence intensities. (C) Western blot showing complement proteins (C3, C5, C4, CFB, MBL2) in each group. The bar graphs indicate the relative intensities of C3 and C5. (D-F) Quantitative polymerase chain reaction analysis of ICAM-1 (D), VCAM-1 (E), and PAI-1 (F). The results are representative of three experiments. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, NS: not statistically significant. Ctrl: control; ICAM-1: intercellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; PAI-1: plasminogen activator inhibitor-1.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Knockdown, Control, Clinical Proteomics, Fluorescence, Western Blot, Real-time Polymerase Chain Reaction

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: I n vivo KLF4 overexpression attenuates transplant-associated thrombotic microangiopathy pathology. (A) Hemoglobin levels and platelet counts in mKLF4-transfected mice versus vector controls. (B) Representative peripheral blood smears (×100) illustrating fewer schistocytes (arrows) in KLF4-overexpressing mice. Scale bars: 10 µm. (C) Plasma lactate dehydrogenase levels in the indicated groups. (D) Representative hematoxylin & eosin and periodic acid-Schiff staining showing glomerular structural improvement in mKLF4-transfected mice versus vector controls. Scale bar: 10 μm. The hashtag (#) indicates glomerular congestion; the asterisk (•) indicates endothelial swelling/detachment. (E) Immunofluorescent detection of C3 and C5b-9 in renal tissue with Hoechst counterstain. The bar graphs show mean fluorescence intensities. Scale bars: 10 μm. (F) Kaplan-Meier survival curves showing significantly improved survival in KLF4-overexpressing mice. The results are representative of three experiments and six mice/group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, NS: not statistically significant. HGB: hemoglobin; Ctrl: control; DMOG: dimethyloxalylglycine; PLT: platelet; LDH: lactate dehydrogenase; H&E: hematoxylin & eosin; PAS: periodic acid-Schiff; DAPI: 4’,6-diamidino-2-phenylindole.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Over Expression, Transfection, Plasmid Preparation, Clinical Proteomics, Staining, Fluorescence, Control

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: Statin treatment ameliorates transplant-associated thrombotic microangiopathy by upregulating KLF4. (A) Hemoglobin and platelet counts in mice treated with pravastatin or vehicle. (B) Peripheral blood smears (×100) showing fewer schistocytes (arrows) in pravastatin-treated mice. Scale bars: 10 μm. (C) Plasma lactate dehydrogenase in each group. (D) Representative staining of renal tissue with hematoxylin & eosin and periodic acid Schiff demonstrating restored glomerular histology in mice treated with pravastatin (scale bar, 10 μm). The hashtag (#) indicates glomerular congestion; the asterisk (•) indicates endothelial swelling/detachment. (E) Immunofluorescent staining of C3 and C5b-9 with Hoechst counterstain. The bar graphs show mean fluorescence intensities. Scale bars: 10 μm. (F) Kaplan–Meier survival curves for mice receiving pravastatin versus vehicle. The results are representative of three experiments and six mice/group. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, NS: not statistically significant. HGB: hemoglobin; Ctrl: control; DMOG: dimethyloxalylglycine; PLT: platelet; LDH: lactate dehydrogenase; DAPI: 4’,6-diamidino-2-phenylindole; H&E: hematoxylin & eosin; PAS: periodic acid-Schiff.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques: Clinical Proteomics, Staining, Fluorescence, Control

Journal: Haematologica

Article Title: KLF4 overexpression protects against complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy

doi: 10.3324/haematol.2025.287676

Figure Lengend Snippet: Schematic diagram of KLF4 as a key negative regulator of complement-mediated endothelial injury in transplant-associated thrombotic microangiopathy. KLF4: Krüppel-like factor 4; ICAM-1: intercellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; PAI-1: plasminogen activator inhibitor-1; CD46: cluster of differentiation 46; sC5b-9: soluble terminal complement complex; C3b: complement 3b fragment; TA-TMA: transplant-associated thrombotic microangiopathy.

Article Snippet: Next, to assess the therapeutic potential of KLF4, we pretreated HUVEC with the KLF4 stimulator APTO253 (MCE, #HY-16291) for 24 h. Flow cytometry showed significant elevations of C3 ( Online Supplementary Figure S2B ) and C5b-9 ( Online Supplementary Figure S2C ) in HUVEC incubated with TA-TMA plasma (termed “TMA plasma” in figures) versus control plasma (“Ctrl plasma” in figures).

Techniques:

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: 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: bioRxiv

Article Title: Circular RNA Circ-Cdr1as modulates Macrophage phenotype and Cardiac Reparative Function by Circ-Cdr1as-miR-7-Klf4 pathway

doi: 10.1101/2025.02.21.639391

Figure Lengend Snippet: (A) qRT-qPCR of KLF4 expression in 3D post-MI left ventricular (LV) tissue compared to sham, normalized to 18S. N=3/group. (Unpaired T-test) (B) RT-qPCR of KLF4 expression in 3D post-MI left ventricular tissue in AAV9-GFP and AAV9-circ-cdr1as groups, normalized to 18S. N=4 (Unpaired-t-test). (C) Changes in KLF4 expression in naïve, pro-inflammatory, and anti-inflammatory macrophages overexpressing circ-cdr1as or knockdown of circ-cdr1as, normalized to 18S. N= 3/group (One-way ANOVA). (D) Changes in KLF4 expression in naïve, pro-inflammatory, and anti-inflammatory macrophages treated with miR-7b-5p mimic or anti-mir-7b-5p, normalized to miR-24. N= 3/group (One-way ANOVA). (E) FACS analysis of F4/80/CD86 + cells (pro-inflammatory MΦ marker) or (F) F4/80/CD206+ cells (anti-inflammatory MΦ marker) in naïve macrophages treated with lentivirus KFL4 or shRNA KLF4 and their respective controls. N=3-4/group (One-way ANOVA). Data are mean ± SEM. NS, non-significant, *p<0.05, ** p<0.01, *** p<0.001. MΦ, macrophages; pro-inflammatory marker: CD86; anti-inflammatory marker: CD206.

Article Snippet: For overexpression of Klf4, BMDMs were transfected with Klf4 (NM_010637) Mouse Tagged ORF Clone Lentiviral Particle (Origene) (titer concentration 1.7×10^7 TU/mL).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Marker, shRNA

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: Journal of Cellular and Molecular Medicine

Article Title: SNHG1 , a KLF4 ‐upregulated gene, promotes glioma cell survival and tumorigenesis under endoplasmic reticulum stress by upregulating BIRC3 expression

doi: 10.1111/jcmm.17779

Figure Lengend Snippet: KLF4 transcriptionally upregulates SNHG1 under ER stress. (A) U251 and T98G cells were treated with 3 μM TM, and the expression levels of KLF4 were detected by Western blot analysis. (B–E) U251 and T98G cells with or without KLF4 knockdown were treated with 3 μM TM for the indicated times. The expression levels of SNHG1 were measured by qRT–PCR, and the protein levels of KLF4 were detected by Western blot analysis. (F) T98G cells with or without SNHG1 overexpression were treated with 3 μM TM for the indicated times, and the expression levels of SNHG1 were measured by qRT–PCR. (G) Schematic illustration of pGL3‐based reporter constructs used in luciferase assays to examine the transcriptional activity of SNHG1. (H) P1, P2 and P3 were transfected into 293T cells with or without KLF4 expression followed by measurement of luciferase activity. (I–J) P3 was transfected into U251 and T98G cells with or without KLF4 knockdown followed by measurement of the luciferase activity of P2. (K) Schematic illustration of the KLF4 wild‐type binding site (BS) and the matching mutant (BSM) used in the luciferase assays. (L) The wild‐type promoter (BS) or the matching mutant (BSM) was transfected into 293T cells with or without KLF4 overexpression followed by measurement of luciferase activity. (M) The wild‐type promoter (BS) or the matching mutant (BSM) was transfected into U251 cells with or without 3 μM TM treatment followed by measurement of luciferase activity. (M–O) ChIP analysis showing the binding of KLF4 to the promoter of SNHG1 in U251 cells with or without KLF4 knockdown or 3 μM TM treatment for the indicated times. Isotype‐matched IgG was used as a negative control. Data in (C), (E), (F), (H), (I), (J), (L) and (M) were analysed by Student's t ‐test (** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used in the present study: GAPDH (Santa Cruz Biotechnology; SC‐25778, 1:1000), PARP (Santa Cruz Biotechnology, SC‐8007, 1:1000), GRP78 (Santa Cruz Biotechnology, SC‐13968, 1:1000 for WB), KLF4 (Cell Signaling Technology, #12173S, 1:500) and BIRC3 (Proteintech, 24304‐1‐AP, 1:1000).

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Over Expression, Construct, Luciferase, Activity Assay, Transfection, Binding Assay, Mutagenesis, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: SNHG1 , a KLF4 ‐upregulated gene, promotes glioma cell survival and tumorigenesis under endoplasmic reticulum stress by upregulating BIRC3 expression

doi: 10.1111/jcmm.17779

Figure Lengend Snippet: KLF4 promotes glioma cell survival, invasion and BIRC3 expression by regulating SNHG1 in response to ER stress treatment. (A–D) SNHG1 was overexpressed in U251 and T98G cells with or without KLF4 knockdown followed by treatment with 3 μM TM. The expression levels of BIRC3 were analysed by Western blot and qRT–PCR analysis. Numbers represent the relative intensities of Western blot bands of BIRC3 to GAPDH. (E–H) Cell apoptosis and viability were detected by Western blot analysis and CCK8 assays. (I–L) Cell invasion was detected by a Transwell assay. Data in (B), (D), (F), (H), (J) and (L) were analysed by Student's t ‐test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: The following antibodies were used in the present study: GAPDH (Santa Cruz Biotechnology; SC‐25778, 1:1000), PARP (Santa Cruz Biotechnology, SC‐8007, 1:1000), GRP78 (Santa Cruz Biotechnology, SC‐13968, 1:1000 for WB), KLF4 (Cell Signaling Technology, #12173S, 1:500) and BIRC3 (Proteintech, 24304‐1‐AP, 1:1000).

Techniques: Expressing, Knockdown, Western Blot, Quantitative RT-PCR, Transwell Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: SNHG1 , a KLF4 ‐upregulated gene, promotes glioma cell survival and tumorigenesis under endoplasmic reticulum stress by upregulating BIRC3 expression

doi: 10.1111/jcmm.17779

Figure Lengend Snippet: Schematic diagram of the mechanism by which SNHG1 promotes the adaptation to ER stress. SNHG1 is a KLF4‐regulated lncRNA that suppresses ER stress‐induced apoptosis and facilitates gliomagenesis by elevating BIRC3 expression.

Article Snippet: The following antibodies were used in the present study: GAPDH (Santa Cruz Biotechnology; SC‐25778, 1:1000), PARP (Santa Cruz Biotechnology, SC‐8007, 1:1000), GRP78 (Santa Cruz Biotechnology, SC‐13968, 1:1000 for WB), KLF4 (Cell Signaling Technology, #12173S, 1:500) and BIRC3 (Proteintech, 24304‐1‐AP, 1:1000).

Techniques: Expressing

Journal: Journal of neuroinflammation

Article Title: The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke.

doi: 10.1186/s12974-023-02742-9

Figure Lengend Snippet: Fig. 2 Co-expression of KLF4-C3 or KLF4-S100A10 after focal cerebral ischemia. A Images show the dual-IF staining for KLF4 with C3 as well as S100A10 in ischemic penumbra from mice 2, 4 and 7 days after focal cerebral ischemia. Scale bar = 100 µm for the left three columns, Scale bar = 25 µm for Zoom panels. B, C Quantification of KLF4 + C3 + cell number (B) and KLF4 + S100A10 + cell number (C) in the ischemic penumbra. Results are expressed as the mean ± standard deviation of the double-positive events per field of view, and the data were analyzed by one-way ANOVA (n = 8 per experimental group). Note that in the ischemic penumbra, the co-expression of KLF4-C3 or KLF4-S100A10 increased with time and reached a maximum level at day 7 post-ischemia. However, the distribution patterns of their expressions were different, where the high levels of KLF4 was expressed, relatively low levels of C3 were expressed in the ischemic penumbra, and vice versa. However, KLF4 was always co-stained well with S100A10 in the penumbra at days 2, 4 and 7 post-ischemia, especially at day 7, KLF4 co-localized with S100A10 extensively. **P < 0.01, ***P < 0.001 compared with 2-day post-ischemia

Article Snippet: Rabbit anti KLF4 polyclonal antibody (11880-1-AP, 1:1000) was gotten from Proteintech (Rosemont, IL).

Techniques: Expressing, Staining, Standard Deviation

Journal: Journal of neuroinflammation

Article Title: The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke.

doi: 10.1186/s12974-023-02742-9

Figure Lengend Snippet: Fig. 3 Influence of silencing of KLF4 on activation of A1/A2 astrocytes under OGD/R conditions. A Representative images of western blot for KLF4, C3, S100A10, TNF-α, iNOS, p-NF-kB in astrocytes transfected with the negative control siRNA (si-Ctl) or siRNA-KLF4 (si-KLF4) at 48 h restoration from OGD. The NO-OGD/R si-Ctl treated cells served as a control. B–G Bar graphs show the quantitative analyses of western blots as ratios of KLF4/β-actin (B), C3/β-actin (C), S100A10/β-actin (D), TNF-α/β-actin (E), iNOS/β-actin (F) and p-NF-kB/total NF-kB (G), and the data were analyzed by two-way ANOVA (n = 4 per experimental group). Note that the protein levels of C3, TNF-α, iNOS and the phosphorylation of NF-κB were significantly elevated relative to the si-Ctl-treated group at 48 h restoration from OGD, but the levels of KLF4 and S100A10 in the si-KLF4-treated astrocytes were markedly reduced. *P < 0.05, **P < 0.01, ***P < 0.001. H, I Representative images of immunofluorescent staining for C3/S100A10, GFAP, and DAPI in astrocytes after indicated treatments. Scale bar = 100 μm. J–O mRNA level of pro- or anti-inflammatory genes was determined by qPCR in the astrocytes of si-Ctl and si-KLF4 group at 48 h restoration of OGD or NO-OGD/R, and the data were analyzed by two-way ANOVA (n = 4 per experimental group). NO-OGD/R si-Ctl-treated cells served as control. Note that the mRNA levels of pro-inflammatory genes including IL-1β (J), TNF-α (K) and iNOS (L) were shown to significantly increase, but anti-inflammatory genes including IL-1ra (M), IL-10 (N), and Arg1 (O) were found to remarkably decrease in astrocytes after OGD/R injury. Furthermore, OGD/R-induced expression of pro-inflammatory genes was exaggerated by diminishing the expression of KLF4 in astrocytes; likewise, the decreased expression of anti-inflammatory genes caused by OGD/R was further augmented by silencing the levels of KLF4 in the astrocytes. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Article Snippet: Rabbit anti KLF4 polyclonal antibody (11880-1-AP, 1:1000) was gotten from Proteintech (Rosemont, IL).

Techniques: Activation Assay, Western Blot, Transfection, Negative Control, Control, Phospho-proteomics, Staining, Expressing

Journal: Journal of neuroinflammation

Article Title: The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke.

doi: 10.1186/s12974-023-02742-9

Figure Lengend Snippet: Fig. 4 Impact of overexpression of KLF4 on activation of A1/A2 astrocytes under OGD/R conditions. A Representative images of western blot for KLF4, C3, S100A10, TNF-α, iNOS, p-NF-kB in astrocytes in astrocytes transfected with control plasmid (mock) or KLF4 overexpression plasmid at 48 h restoration from OGD. The NO-OGD/R mock treated cells served as a control. B–G Bar graphs show the quantitative analyses of western blots as ratios of KLF4/β-actin (B), C3/β-actin (C), S100A10/β-actin (D), TNF-α/β-actin (E), iNOS/β-actin (F), and p-NF-kB/total NF-kB (G), and the data were analyzed by two-way ANOVA (n = 4 per experimental group). Note that compared to the mock-transfected group, the protein levels of C3, TNF-α, iNOS and the phosphorylation of NF-κB were markedly reduced, but the levels of KLF4 and S100A10 were significantly elevated in astrocytes transfected with KLF4 after 48 h restoration from OGD. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. H, I Representative images of immunofluorescent staining for C3/S100A10, GFAP and DAPI in astrocytes transfected with mock or KLF4 overexpression plasmid after OGD/R treatment. Scale bar = 100 μm. J–O mRNA level of pro- or anti-inflammatory genes in the astrocytes were determined by qPCR in the mock or KLF4 overexpression group at 48 h restoration of OGD or NO-OGD/R, and the data were analyzed by two-way ANOVA (n = 4 per experimental group). NO-OGD/R mock-treated cells served as control. Note that compared to the mock-transfected group, overexpression of KLF4 decreased the expression of IL-1β (J), TNF-α (K), and iNOS (L), but increased the levels of IL-1ra (M), IL-10 (N), and Arg1 (O) in astrocytes under OGD/R conditions. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant

Article Snippet: Rabbit anti KLF4 polyclonal antibody (11880-1-AP, 1:1000) was gotten from Proteintech (Rosemont, IL).

Techniques: Over Expression, Activation Assay, Western Blot, Transfection, Control, Plasmid Preparation, Phospho-proteomics, Staining, Expressing

Journal: Journal of neuroinflammation

Article Title: The critical role of KLF4 in regulating the activation of A1/A2 reactive astrocytes following ischemic stroke.

doi: 10.1186/s12974-023-02742-9

Figure Lengend Snippet: Fig. 5 Schematic diagram showing astrocytic KLF4 regulating the activation of A1/A2 reactive astrocytes following ischemic stroke. After stroke onset, astrocytes are excessively activated. The classically activated astrocytes (A1 subtype) exert neurotoxic effects by releasing pro-inflammatory mediators, such as iNOS, TNF-α and IL-1β, while alternatively activated astrocytes (A2 subtype) perform neuroprotective effects by secreting anti-inflammatory mediators, such as Arg1, IL-1ra and IL-10. Of interest, astrocytic KLF4 inhibited activation of C3 positive A1 astrocytes but promoted S100A10 positive A2 astrocytes polarization following ischemic stroke by modulating expressions of nuclear factor-kB

Article Snippet: Rabbit anti KLF4 polyclonal antibody (11880-1-AP, 1:1000) was gotten from Proteintech (Rosemont, IL).

Techniques: Activation Assay