Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: KLF5 protein expression and Progression-free survival curves in EOC. (A) A significant difference in expression levels was noted between normal ovary ( n = 45) and EOC tissues ( n = 425) ( p < 0.0001). (B) Immunohistochemical analysis of KLF5 and pSTAT-3 expression in EOC TMA. An EOC array spot showing overexpression of KLF5 (a) and pSTAT-3 (C) . In contrast, another EOC tissue array spots showing low expression of KLF5 (b) and pSTAT-3 (D) . 20X/0.70 objective on an Olympus BX 51 microscope. (Olympus America Inc, Center Valley, PA, USA) with the inset showing a 40X 0.85 aperture magnified view of the same TMA spot. (C) Kaplan-Meier survival analysis for the prognostic significance of KLF5 expression in EOC showed that patients with overexpression of KLF5 had reduced progression-free survival at 5 years compared to tumors showing low expression of KLF5 ( p = 0.0182). (D) EOC patients with co-expression of KLF5 and p-STAT3 had reduced progression-free survival at 5 years compared to tumors without co-expression of KLF5 and p-STAT3 ( p = 0.0034).

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: Expressing, Immunohistochemical staining, Over Expression, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: Clinicopathological associations of KLF5 protein expression in EOC.

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: KLF5 drives STAT3-activation in EOC. (A) Basal expression of KLF5 and pSTAT3 in EOC cell lines. Proteins were isolated from six EOC cell lines and immunoblotted with antibodies against KLF5, pSTAT3, STAT3 and GAPDH. (B) Silencing of KLF5 inhibits STAT3 activation. EOC cells were transfected with scrambled siRNA and KLF5 siRNA (50 and 100 nM). After 48 hours, cells were lysed and proteins were immunoblotted with antibodies against KLF5, pSTAT3, STAT3 and GAPDH. (C) ML264 treatment down-regulates KLF5 expression and STAT3 activation in EOC cells. EOC cells were treated with indicated doses of ML264 for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immuno-blotted with antibodies against KLF5, pSTAT3, STAT3 and GAPDH as indicated. (D) Knockdown of STAT3 has no effect on KLF5 expression. EOC cells were transfected with scrambled siRNA and STAT3 siRNA (50 and 100 nM). After 48 h, cells were lysed and proteins were immunoblotted with antibodies against pSTAT3, STAT3, KLF5 and GAPDH. (E) Forced expression of KLF5 increases STAT3 activation. MDAH2774 cells were transfected with either empty vector or KLF5 cDNA for 48 hours. Proteins were isolated and immunoblotted with antibodies against KLF5, pSTAT3, STAT3 and GAPDH for equal loading. All the experiments were repeated for at least two times with the same results.

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: Activation Assay, Expressing, Isolation, Transfection, Lysis, SDS Page, Membrane, Knockdown, Plasmid Preparation

Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: Inhibition of KLF5 decreases invasion, migration and progression of epithelial-to-mesenchymal transition in EOC cells. (A , B) KLF5 inhibition decreases the invasive capacity of EOC cells. EOC cells were pre-treated with universal caspase inhibitor, z-VAD/fmk (80 µM) for 3 h and subsequently treated with indicated doses of ML264 and seeded into the upper compartment of invasion chambers. The bottom chambers were filled with RPMI media. After 24 h incubation, invaded cells were fixed, stained and quantified. (C) KLF5 inhibition causes reduction in the migration capacity of EOC cells. EOC cells were pre-treated with universal caspase inhibitor, z-VAD/fmk (80 µM) for 3 h and subsequently treated with indicated doses of ML264 and seeded into the upper compartment of migration chambers. The bottom chambers were filled with RPMI media. After 24 h incubation, migrated cells were fixed, stained and quantified. (D) ML264 treatment down-regulates the expression of EMT markers in EOC cells. EOC cells were treated with indicated doses of ML264 for 48 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to immobilon membrane, and immuno-blotted with antibodies against KLF5, pSTAT3, STAT3, E-cadherin, N-cadherin, Vimentin, Twist, MMP-2, MMP-9 and GAPDH as indicated. (E) Silencing of KLF5 down-regulates the expression of EMT markers in EOC cells. EOC cells were transfected with scrambled siRNA and KLF5 siRNA (50 and 100 nM). After 48 h, cells were lysed and proteins were immunoblotted with antibodies against KLF5, pSTAT3, STAT3, E-cadherin, N-cadherin, Vimentin, Twist, MMP-2, MMP-9 and GAPDH. Data presented in the bar graphs are the mean ± SD of triplicates in an independent experiments which was repeated for at least two times with the same results. *Indicates a statistically significant difference compared to control with p < 0.05. Western blot experiments were repeated at least two times with the same results.

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: Inhibition, Migration, Incubation, Staining, Expressing, Lysis, SDS Page, Membrane, Transfection, Control, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: Downregulation of KLF5 inhibits EOC cell growth in vitro. (A) ML264 inhibits cell viability. EOC cells (10 4 ) were incubated with indicated doses of ML264 for 48 hours. Cell viability was performed using MTT. (B, C) ML264 inhibited clonogenicity. EOC cells (8 × 10 2 ) after ML264 treatment were seeded into each of two dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet and colonies were counted. (D, E) Knockdown of KLF5 decreases clonogenicity. EOC cells were transfected with scrambled siRNA and KLF5 siRNA (50 and 100 nM). After 48 h, cells (8 × 10 2 ) were seeded into each of two dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet and colonies were counted. (F-G) Forced expression of KLF5 increases clonogenicity. MDAH2774 cells were transfected with either empty vector or KLF5 cDNA. After 48 h, cells (8 × 10 2 ) were seeded into each of two dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet and colonies were counted. (H) ML264 induces apoptosis in EOC cell lines. EOC cells were treated with indicated doses of ML264 for 48 h and cells were stained with fluorescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry. Data presented in the bar graphs are the mean ± SD of triplicates in an independent experiments which was repeated for at least two times with the same results. *Indicates a statistically significant difference compared to control with p < 0.05.

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: In Vitro, Incubation, Staining, Knockdown, Transfection, Expressing, Plasmid Preparation, Flow Cytometry, Control

Journal: Frontiers in Pharmacology

Article Title: Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer

doi: 10.3389/fphar.2020.598880

Figure Lengend Snippet: Inhibition of KLF5 decreases spheroid growth in EOC cells. (A, B) Isolation of spheroid-forming cells from EOC cells. Sphere forming assay was performed by culturing EOC cells (5 × 10 2 cells/well) in sphere medium for 14 days in 24-well ultra-low attachment plates. Proteins were isolated from spheroid-forming cells and respective parental adherent cells and immunoblotted with antibodies against KLF5, pSTAT3, STAT3, CD44, CD133, NANOG, OCT4 and GAPDH. (B, C) Silencing of KLF5 inhibits self-renewal ability of spheroids. EOC cells were transfected with KLF5 shRNA and cells were subjected to sphere forming assay. Spheroids in the entire well were counted. (D) Silencing of KLF5 inhibits stemness of spheroids as confirmed by immunoblotting using stem cell markers. EOC cells were transfected with scramble or KLF5 shRNA’s and grown in sphere medium. Proteins were isolated from spheroids and immunoblotted with antibodies against KLF5, pSTAT3, STAT3, CD44, CD133, NANOG, OCT4 and GAPDH. Data presented in the bar graphs are the mean ± SD of triplicates in an independent experiments which was repeated for at least two times with the same results. *Indicates a statistically significant difference compared to control with p < 0.05. Western blot experiments were repeated at least two times with the same results.

Article Snippet: Plasmid DNA encoding human KLF5 (RC202438) and shRNA targeting human KLF5 (TR311886) were purchased from Origene (Rockville, MD).

Techniques: Inhibition, Isolation, Transfection, shRNA, Western Blot, Control

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: iMDP-3 cells were cultured in DM (DMEM supplemented with 10% FBS, antibiotics, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate and 100 nM dexamethasone) for 0, 1, 3, 5, 7, 11 and 14 days. ( a ) Alizarin Red S (ARS) and ( b ) Alkaline phosphatase (ALP) staining of iMDP-3 cells on days 7 and 14 after differentiation induction by low and high magnifications. ( c ) Cell numbers on days 7 and 14 after differentiation induction. ( d ) Expression of Klf5 protein was detected by Western blot analysis using antibodies specific to Klf5 and β-actin. Protein expression of Klf5 was upregulated during odontoblastic differentiation of iMDP-3 cells. ( e ) Protein expression of Klf5 and β-actin was quantitated using image J software. Expression of Klf5 was normalized to β-actin expression. Expression level of Klf5 proteins on day 0 acts as one-fold increase. ( f ) Klf5, ( g ) Dspp and ( h ) Dmp1 mRNA expression was followed by qRT-PCR relative to Cyclo A. mRNA expression of Klf5, Dspp and Dmp1 is upregulated during odontoblastic differentiation of iMDP-3 cells. Expression level of Klf5, Dsp and Dmp1 at different time periods was divided by the Klf5, Dsp and Dmp1 expressions on day 0. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Cell Culture, Staining, Expressing, Western Blot, Software, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a–e ) in iMDP-3 cells, ( f–j ) in MD10-F2 cells, the coexpression of Klf5 ( green , b and g) with Dsp ( red , c and h) was observed in these cells. ( k–o ) in molar at PN2, ( p–t ) in molar at PN6, Klf5 ( green , l and q) expression was observed in ameloblasts, SI, HERS, odontoblasts and dentin. Expression of Dsp ( red , m and r) was also overlapped with that of Klf5. a,f,k and p show bright images. ( d,i,n and s ) Cells were stained with Hoechst for nuclei. e , j , o and t are merged. Similar to the above description, (a′–t′) in incisors at PN3, coexpression of Klf5 ( green , e′–h′) and Dsp ( red , i′–l′) was visible in odontoblasts. a′–d′ show bright images. (m′–p′) Cell nuclei were stained with Hoechst. (q′–t′) The images were merged. Bars, 20 μm ( a–t ), 10 μm ( k–t , a′–t′).

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a–e ) in iMDP-3 cells, ( f–j ) in MD10-F2 cells, the coexpression of Klf5 ( green , b and g) with Dmp1 ( red , c and h) was observed in these cells. ( k–o ) in molar at PN2, ( p–t ) in molar at PN6, Klf5 ( green, l and q) expression was observed in ameloblasts, odontoblasts and dentin. Expression of Dmp1 ( red, m and r) was also overlapped with that of Klf5. a,f,k and p show bright images. ( e, j, o and t ) Images were merged. ( d , i , n and s ) Cells were stained with Hoechst for nuclei. Similar to the above description, (a′)–(t′) in incisors at PN3, coexpression of Klf5 ( green , e′–h′) and Dmp1 ( red , i′-l′) was visible in odontoblasts. a′–d′ show bright images. (m′–p′) Cell nuclei were stained with Hoechst. (q′–t′) The images were merged. Bars 20 μm ( a–t ), 10 μm ( a–t , a′–t′).

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Expressing, Staining

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Klf5 gene plasmid tagged with green fluorescent protein (GFP) was overexpressed by transient transfection in iMDP-3 cells. After 48 h transfection, more than 50% of cells were positive to GFP. Klf5 overexpression in iMDP-3 cells was examined using RT-PCR ( b ) and Western blot analyses ( e and f ). The Dspp mRNA ( c ) and Dsp protein ( e and g ) levels were all increased on 48 h in Klf5 overexpression cells compared with control group. The mRNA ( d ) and protein ( e and h ) levels of Dmp1 were also increased on 48 h in Klf5 overexpression cells compared with control group. ( i and j ) Expression of ALP mRNA and protein was significantly increased after Klf5 overexpression in iMDP-3 cells. ARS ( k ) assay were used to monitor the progress of mineralization in Klf5 overexpression cells as compared to control groups. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

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

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: iMDP-3 cells were transfected with different Dspp and Dmp1 promoter constructs with either pcDNA3-Klf5 or pcDNA3 plasmid as control. Reporter activities were measured by a dual-luciferase assay in the presence or absence of pcDNA3-Klf5 co-transfection. ( a ) Activation of Dspp promoters containing pGL3-5.7 kb, pGL3-2.6 kb, pGL3-1.5 kb, pGL3-1,318 bp and pGL3-591bp was increased 5.8-, 3.0-, 1.8-, 1.9- and 1.1-folds, respectively. ( b ) Effect of different Klf5 concentrations on Dspp promoter activity containing pGL3-5.7 kb. ( c ) The transcription activities appeared to increase in a dosage-dependent manner for Dspp promoter-5.7 kb with Klf5 transfection. ( d ) Activation of Dmp1 promoters containing pGL3-2.6 kb, pGL3-1,656 bp, pGL3-1,187 bp, pGL3-656bp and pGL3-213bp were increased 5.4-, 4.0-, 3.2-, 2.2- and 2.0-folds, respectively. ( e ) Effect of different Klf5 concentrations on Dmp1 promoter activity containing pGL3-2.6 kb. ( f ) The transcription activities appeared to increase in a dosage-dependent manner for Dmp1 promoter-2.6 kb with Klf5 transfection. Luciferase (Luc) activity was normalized to the control group. * P < 0.05; ** P < 0.01.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Transfection, Construct, Plasmid Preparation, Luciferase, Cotransfection, Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: Sequences of oligonucleotides of potential Klf5-binding sites.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques:

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Scheme represents of 9 potential Klf5 binding sites in the mouse Dspp intron 1. ( b ) Conserved non-coding sequence islands are found by comparing the proximal promoters and the first introns of the mouse, rat and human Dspp genes. Many of the conserved non-coding sequences are in these regions. The scale on the y-axis goes to from 50 to 100% homology. The pink regions are peaks of conserved nucleotide sequences with a minimum of 70 homologies. ( c ) Nine 32 P-labeled double-stranded Dspp probes were generated for electrophoresis mobility shift assays (EMSAs). These included Dspp-site 1 to Dspp-site 9. EMSAs were carried out using nuclear extracts obtained from iMDP-3 cells. ( c , lane 1 ) free Dspp-site 1 probes only; (c, lane 2 ) binding of nuclear extracts to Dspp-site 1 probe; (c, lane 3 ) the labeled probe with nuclear extracts and 100× cold Dspp-site 1 oligo; (c, lane 4 ) the labeled probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 5 ) the labeled probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 6 ) the labeled probe with nuclear extracts and 2 μg Klf5 antibody; (c, lane 7 ) free Klf consensus sequence probe only; (c, lane 8 ) the binding of nuclear extracts to Klf probe; (c, lane 9 ) Klf probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 10 ) Klf probe with nuclear extracts and 100× cold Dspp-site 1 oligo; (c, lane 11 ) Klf probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 12 ) Klf probe with nuclear extracts and 2 μg Klf5 antibody. (d, lane 1–12 ) Dspp-site 2 and Klf probes; (e, lane 1–12 ) Dspp-site 3 and Klf probes; (f, lane 1–12 ) Dspp-site 4 and Klf probes; (g, lane 1–12 ) Dspp-site 5 and Klf probes; (h, lane 1–12 ) Dspp-site 6 and Klf probes; (i, lane 1–12 ) Dspp-site 7 and Klf probes; (j, lane 1–12 ) Dspp-site 8 and Klf probes; (k, lane 1–12 ) Dspp-site 9 and Klf probes.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Sequencing, Labeling, Generated, Electrophoresis, Mobility Shift

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) Scheme represents of 2 potential Klf5 binding sites in human Dmp1 promoter. ( b ) Conserved non-coding sequences of Dmp1 gene promoters in three mammalian species, mouse, rat and human are retrieved using Vista plots for comparative genomic analysis of gene promoters. The pink regions represent peaks of conserved nucleotide sequences with a minimum of 70 homologies. Many of the conserved non-coding sequences are in these regions. The scale on the y-axis goes to from 50 to 100% homology. Two 32 P-labeled double-stranded Dmp1 probes were generated for EMSAs. These included Dmp1-site 1 and Dmp1-site 2. EMSAs were carried out using nuclear extracts obtained from iMDP-3 cells. (c, lane 1 ) free Dmp1-site 1 probes only; (c, lane 2 ) the binding of nuclear extracts to Dmp1-site 1 probe; (c, lane 3 ) the probe with nuclear extracts and 100× cold Dmp1-site 1 oligo; (c, lane 4 ) the probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 5 ) the probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 6 ) the probe with nuclear extracts and 2 μg Klf5 antibody; (c, lane 7 ) free Klf consensus sequence probe only; (c, lane 8 ) the binding of nuclear extracts to the Klf probe; (c, lane 9 ) the probe with nuclear extracts and 100× cold Klf consensus sequence oligo; (c, lane 10 ) the probe with nuclear extracts and 100× cold Dmp1-site 1 oligo; (c, lane 11 ) the probe with nuclear extracts and 1 μg Klf5 antibody; (c, lane 12 ) the probe with nuclear extracts and 2 μg Klf5 antibody. (d, lane 1–12 ) Dmp1-site 2 and Klf probes.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Labeling, Generated, Sequencing

Journal: Scientific Reports

Article Title: Klf5 Mediates Odontoblastic Differentiation through Regulating Dentin-Specific Extracellular Matrix Gene Expression during Mouse Tooth Development

doi: 10.1038/srep46746

Figure Lengend Snippet: ( a ) The diagram shows that six pairs of primers were designed to amplify the Klf5 binding sites in the first intron 1 of mouse Dspp gene from Dspp-site 1 to Dspp-site 9 in vivo for ChIP assay. The position number was stated as Dspp-primer 1: 50 bp to 257 bp; Dspp-primer 2: 1,219 bp to 1,422 bp; Dspp-primer 3: 1,885 bp to 2,074 bp; Dspp-primer 4: 2,177 bp to 2,468 bp; Dspp-primer 5: 2,574 bp to 2,861 bp; Dspp-primer 6: 3,025 bp to 3,265 bp. ( b–g ) ChIP assay showed that endogenous Klf 5 interacted with its motifs in the Dspp regulatory regions while Klf 5 overexpression significantly increased binding to its motifs in Dspp regulatory regions from Dspp site 1 to Dspp site 9 in vivo . ( h ). Two pairs of primers were used for the Klf5 binding sites in human Dmp1 promoter from Dmp1-site 1 to Dmp1-site 2 for ChIP assay. ChIP assay was performed with chromatin from iMDP-3 cells with transfection of Dspp-5.7 kb reporter construct ( b–g ) and Dmp1–2.6 kb promoter construct (i and j) with either pcDNA3-Klf5 or pcDNA3 plasmid. The results revealed that Klf5 binds to the Dspp regulatory regions encompassing the CACCC/GGGTG boxes between +73 to +2.8 kb and the Dmp1 promoter between −2.6 kb to −1,656 bp in vivo and exhibited an increasing transcription in Klf5-stimulated groups in iMDP-3 cells.

Article Snippet: The plasmid of Klf5 (pcDNA3-Klf5-GFP) was obtained from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, In Vivo, Over Expression, Transfection, Construct, Plasmid Preparation

Journal: BMC medicine

Article Title: Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

doi: 10.1186/s12916-023-02763-4

Figure Lengend Snippet: Fig. 1 Screening of FDA-approved drugs for those that inhibit cellular invasion induced by acetylated KLF5. a Schematic of the screening workflow. b Inhibitory effects of 1987 FDA-approved drugs, along with vehicle control (green dots pointed by green arrows), on the invasion of PC-3-KQ cells, as analyzed using the 3D spheroid invasion assay. Each dot represents a drug or control, and the dots are aligned along the X-axis. Y-axis indicates the average invasion area between two well for each drug. The blue horizontal line indicates the mean invasion area of all drugs and controls, while the red horizontal line indicates 50% of the mean invasion area. c Proportions of the 1987 drugs that are approved as non-oncological drugs (purple color), oncological targeted therapeutic drugs (orange color), and oncological chemotherapeutic drugs (red color). d Identification of the most effective 25 drugs by using the 3D spheroid invasion assay with multiple concentrations (μM). The heatmap shows the invasion rates (%) of each drug’s multiple concentrations when compared to the vehicle control, as indicated by blue grids with varying intensities. Names of the 25 drugs are shown at the bottom, with 6 of the most effective ones marked by red boxes. e The chemical structure of nitazoxanide

Article Snippet: After stopping the reaction by adding EDTA, DNA was fragmented by sonication, and the extracts were incubated with KLF5 antibody (Cat #: 21017-1-AP, Proteintech) or IgG (Cat #: 2729P, Cell Signaling Technology).

Techniques: Control, Invasion Assay

Journal: BMC medicine

Article Title: Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

doi: 10.1186/s12916-023-02763-4

Figure Lengend Snippet: Fig. 5 Nitazoxanide reverses the effects of Ac-KLF5 on the expression of many genes in PC-3 cells. a Volcano plots of all quantified genes from the gene expression profiles between the KLF5K369Q and KLF5K369R in PC-3 cells. b Scatter plot of differentially expressed genes (DEGs) between PC-3-KQ cells with and without NTZ treatment and DEGs between PC-3-KQ and PC-3-KR cells. Dots in the upper left quadrant represent genes that were downregulated by KLF5K369Q but upregulated by NTZ, whereas dots in the lower right quadrant indicate genes that were upregulated by KLF5K369Q but downregulated by NTZ in PC-3 cells. An adjusted p value <0.05 was used to define all altered genes. c Heatmap showing DEGs in panel A’s upper left and lower right quadrants. The color bar at the right indicates log2 fold changes (Log2FC)

Article Snippet: After stopping the reaction by adding EDTA, DNA was fragmented by sonication, and the extracts were incubated with KLF5 antibody (Cat #: 21017-1-AP, Proteintech) or IgG (Cat #: 2729P, Cell Signaling Technology).

Techniques: Expressing, Gene Expression

Journal: BMC medicine

Article Title: Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

doi: 10.1186/s12916-023-02763-4

Figure Lengend Snippet: Fig. 6 Association of Ac-KLF5- and NTZ-responsive genes with patient survival. a Higher expression levels of 7 genes correlate with overall survival (OS) in prostate cancer patients, as determined by the Kaplan-Meier analysis using the SU2C dataset. b Expression levels of 7 Ac-KLF5- and NTZ-responsive genes were analyzed using the GSE21034 dataset [29].

Article Snippet: After stopping the reaction by adding EDTA, DNA was fragmented by sonication, and the extracts were incubated with KLF5 antibody (Cat #: 21017-1-AP, Proteintech) or IgG (Cat #: 2729P, Cell Signaling Technology).

Techniques: Expressing

Journal: BMC medicine

Article Title: Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer.

doi: 10.1186/s12916-023-02763-4

Figure Lengend Snippet: Fig. 8 NTZ binds to KLF5 protein regardless of its acetylation status. a Fluorescence intensities of KLF5, KLF5K369Q, and KLF5K369R proteins were measured after incubation with different concentrations of NTZ. b Calculation of the Kd values for dissociation constant using the Origin software and by fitting the fluorescence quenching data. c Circular dichroism (CD) spectra of KLF5, KLF5K369Q, and KLF5K369R incubated with NTZ. The concentrations of both proteins and NTZ are 0.2 μM. d Binding of NTZ to the purified different forms of KLF5 proteins using HPLC analysis

Article Snippet: After stopping the reaction by adding EDTA, DNA was fragmented by sonication, and the extracts were incubated with KLF5 antibody (Cat #: 21017-1-AP, Proteintech) or IgG (Cat #: 2729P, Cell Signaling Technology).

Techniques: Fluorescence, Incubation, Software, Circular Dichroism, Binding Assay, Purification

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 1. Evaluation of KLF5 expression in benign and malignant thyroid lesions. (A) Representative IHC images of KLF5 in non-tumor tissues, thyroid cancer tissues without LN metastasis and thyroid cancer tissues with LN metastasis. (B) Statistical analysis of KLF5 scores in non-tumor tissues, thyroid cancer tissues without LN metastasis and thyroid cancer tissues with LN metastasis. KLF5, Kruppel-like factor 5; LN, lymph node; IHC, immunohistochemistry.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Expressing, Immunohistochemistry

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 2. Effect of KLF5 on cell growth in vitro. (A) Knockdown and overexpression efficacy of KLF5 verified by western blot analysis. (B) Cell proliferation of SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells was evaluated using a CCK-8 assay (upper panel); Cell proliferation of B-CPAP/NC and B-CPAP/KLF5 cells was determined using a CCK-8 assay (lower panel). (C) Anchorage-independent growth assay in SW579 and B-CPAP cells. Knockdown of KLF5 inhibited the anchorage-independent growth of SW579 cells while the overexpression of KLF5 promoted the anchorage-independent growth of B-CPAP cells. KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; OD, optical density; CCK-8, Cell Counting Kit-8.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: In Vitro, Knockdown, Over Expression, Western Blot, CCK-8 Assay, Growth Assay, Small Interfering RNA, Negative Control, Cell Counting

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 3. KLF5 promoted the invasive and metastatic potential of thyroid cancer cells in vitro. (A) Cell migration/invasion of SW579/scrRNA, SW579/ siKLF5#1 and SW579/siKLF5#2 was analyzed using a Transwell assay. (B) Cell migration/invasion of B-CPAP/NC and B-CPAP/KLF5 cells was assessed using a Transwell assay. (C) Western blot analysis of lysates from SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells using anti-E-cadherin, anti- vimentin, anti-Twist1 and anti-fibronectin and (D) the western blot analysis results quantified. (E) Immunostaining of phalloidin (F-actin) in SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2 cells. (F) Western blot analysis of lysates from B-CPAP/NC and B-CPAP/KLF5 cells using the indicated antibodies and (G) the western blot analysis results quantified. (H) Immunostaining of phalloidin (F-actin) in B-CPAP/NC and B-CPAP/KLF5 cells. KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; Twist1, Twist family BHLH transcription factor 1.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: In Vitro, Migration, Transwell Assay, Western Blot, Immunostaining, Small Interfering RNA, Negative Control

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 4. Effect of KLF5 on the ERK pathway and the AKT pathway. Western blot analysis of p-ERK, ERK, p-AKT and AKT in SW579 cells (SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2) and B-CPAP cells (B-CPAP/NC and B-CPAP/KLF5). KLF5, Kruppel-like factor 5; scrRNA, scrambled RNA; si-, small interfering RNA; NC, negative control; AKT, protein kinase B; p-, phosphorylated; ERK, extracellular-regulated kinase; NS, not significant.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Western Blot, Small Interfering RNA, Negative Control

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 5. (A) Western blotting of p-IκB-α, p-IKK-β and nuclear NF-κB p65 in SW579 cells (SW579/scrRNA, SW579/siKLF5#1 and SW579/siKLF5#2, left panel) and B-CPAP cells (B-CPAP/NC and B-CPAP/KLF5, right panel); and p84 was used as a nuclear marker. (B) Quantified western blotting results. (C) Immunofluorescent staining of NF-κB p65 in SW579/scrRNA, SW579/siKLF5#1, SW579/siKLF5#2, B-CPAP/NC and B-CPAP/KLF5 cells. NF-κB, nuclear factor-κB; p-, phosphorylated-; IKK-β, inhibitor of nuclear factor κB kinase subunit β; IκB-α, nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α; scrRNA, scrambled RNA; si, small interfering RNA; NC, negative control; KLF5, Kruppel-like factor 5.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Western Blot, Marker, Staining, Small Interfering RNA, Negative Control

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 6. KLF5 promoted cell growth and metastasis through the NF-κB signalling pathway. B-CPAP/KLF5 cells were treated with the specific NF-κB inhibitor SC75741 (5 µM) and the effect was assessed. (A) B-CPAP/KLF5 cells were treated with SC75741 or DMSO for 48 h. Western blotting of cell lysates from B-CPAP/NC, B-CPAP/KLF5, B-CPAP/KLF5+DMSO and B-CPAP/KLF5+SC75741 cells revealed that the NF-κB inhibitor SC75741 decreased the nuclear NF-κB p65 level of B-CPAP/KLF5 cells; and p84 was used as a nuclear marker. (B) Quantified western blotting results. NF-κB inhibitor SC75741 suppressed the (C) proliferation, (D) anchorage-independent growth and (E) migration/invasion ability of B-CPAP/KLF5 cells. NF-κB, nuclear factor-κB; NC, negative control; KLF5, Kruppel-like factor 5; DMSO, dimethyl sulfoxide.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Western Blot, Marker, Migration, Negative Control

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 7. FBW7 promotes the proteasomal degradation of the KLF5 protein. (A) Interactions between KLF5 and FBW7 were assessed using a co-immuno precipitation assay. Left panel: Cell lysates from SW579 cells were immunoprecipitated with anti-KLF5 or anti-IgG; right panel: Cell lysates from SW579 cells were immunoprecipitated with anti-FBW7 or anti-IgG. (B) Protein half-life of KLF5 was assessed following treatment with CHX (0.1 mg/ml). Additionally, MG132 treatment (5 µM for 8 h) inhibited FBW7‑induced KLF5 degradation in SW579 cells. (C) Quantified western blotting results. FBW7, F-box/WD repeat-containing protein 7; KLF5, Kruppel-like factor 5; IgG, immunoglobulin G; CHX, cycloheximide.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Immunoprecipitation, Western Blot

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 8. Correlation of KLF5 with the expression of FBW7 in the tissues of patients with thyroid cancer. (A) Representative IHC images of KLF5 and FBW7 in thyroid cancer tissues. (B) Correlation between the expression levels of KLF5 and FBW7 in thyroid cancer tissues. (C) Diagram presenting a summary of the results of the present study. It was proposed that the dysregulation of the tumor suppressor FBW7 reduces KLF5 degradation. Then, KLF5 promotes NF-κB p65 nuclear translocation, which in turn promotes the transcriptional levels of multiple downstream genes. FBW7, F-box/WD repeat-containing protein 7; KLF5, Kruppel-like factor 5; IHC, immunohistochemistry; NF-κB, nuclear factor-κB.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: Expressing, Translocation Assay, Immunohistochemistry

Journal: Oncology reports

Article Title: KLF5 promotes the tumorigenesis and metastatic potential of thyroid cancer cells through the NF-κB signaling pathway.

doi: 10.3892/or.2018.6687

Figure Lengend Snippet: Figure 9. KLF5 promoted the in vivo tumor growth and metastasis of thyroid cancer cells. (A) Knockdown efficacy of KLF5 by two shRNAs was confirmed by western blotting. (B) SW579/shNC, SW579/shKLF5#1 and SW579/shKLF5#2 cells were subcutaneously injected into the right flanks of nude mice and (C) the growth curve of tumor volumes was obtained. (D) Mean weight of the tumors from the nude mice of the indicated groups. (E) Representative images of the immunohistochemistry analysis of KLF5, E-cadherin and ki67. (F) B-CPAP/NC and B-CPAP/KLF5 cells were injected intravenously into the tail vein of mice. Representative H&E staining results of lungs are presented. (G) Number of lung metastatic foci in the B-CPAP/NC and B-CPAP/KLF5 groups (5 mice per group). KLF5, Kruppel-like factor 5; shRNA/sh, short hairpin RNA; NC, negative control; H&E, hemotoxylin and eosin.

Article Snippet: To stably silence KLF5, pLVshRNA-EGFP(2A)Puro vectors (Cyagen US Inc., Santa Clara, CA, USA) expressing short hairpin RNA (shRNA/sh) negative control (shNC), shKLF5#1 (target sequence: 5'-CGATTACCCTGGTTGCACA-3') or shKLF5#2 (target sequence: 5'-AAGCTCACCTGAGGACTCA-3') were established.

Techniques: In Vivo, Knockdown, Western Blot, Injection, Immunohistochemistry, Staining, shRNA, Negative Control

Journal: Molecular Cancer Research

Article Title: Up-Regulation of Krüppel-Like Factor 5 in Pancreatic Cancer Is Promoted by Interleukin-1β Signaling and Hypoxia-Inducible Factor-1α

doi: 10.1158/1541-7786.mcr-08-0525

Figure Lengend Snippet: FIGURE 1. Expression and regulation of KLF5 in pancreatic cancer. Expression of KLF5 in KRAS-mutated pancreatic cancer cells (COLO357, HPAF-II, and L3.6pl) and KRAS wild-type cells (BxPC3 and Panc89) were investigated by real-time PCR and Western blotting (at 50% cell density). A. KLF5 mRNA expression was markedly higher in pancreatic cancer cells compared with colon cancer cells (HCT116) harboring a KRAS mutation, which served as a positive control (*, P < 0.05; compared with HCT116). B. Western blot analysis for KLF5 expression and MEK phosphorylation in cancer cells. C. Signaling inhibitors to Erk1/2 (UO126), PI3K/Akt (LY294002), SAPK (SP600125), and p38 (SB203580) were used to identify pathways involved in KLF5 regulation. Changes in KLF5 expression (HPAF-II) were determined after 24 h of incubation with inhibitors by real-time PCR (top) and by Western blotting (bottom; *, P < 0.01, versus control). D. Similarly, in BxPC3 cells, regulation of KLF5 mRNA expression was additionally investigated by using a Raf kinase inhibitor (NVP-AAL881; 20 h; *, P < 0.01; **, P < 0.05, versus control). E. In HCT116 cells, blocking either MAPK/Erk or Raf kinase significantly lowered KLF5 expression (*, P < 0.01). F. Western blot analysis for KLF5 expression in pancreatic (HPAF-II and BxPC3) and colon cancer cells (HCT116) upon inhibition of signaling pathways (20 h) Again, inhibition of MAPK/Erk (UO126) did not lower KLF5 in pancreatic cancer cells but reduced KLF5 in HCT116 cells. Columns, mean; bars, SEM.

Article Snippet: The anti-KLF5 antibody was obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Mutagenesis, Positive Control, Phospho-proteomics, Incubation, Control, Blocking Assay, Inhibition, Protein-Protein interactions

Journal: Molecular Cancer Research

Article Title: Up-Regulation of Krüppel-Like Factor 5 in Pancreatic Cancer Is Promoted by Interleukin-1β Signaling and Hypoxia-Inducible Factor-1α

doi: 10.1158/1541-7786.mcr-08-0525

Figure Lengend Snippet: FIGURE 2. Effect of interleukin-1β on KLF5 expression. A. Western blot analysis of IL-1β (10 ng/mL)–mediated signaling in HPAF-II pancreatic cancer cells. Stimulation with IL-1β activated multiple signaling pathways, including Erk, Akt, SAPK, and p38. B. The effect of IL-1β (10 ng/mL; 24 h) on KLF5 mRNA expression (HPAF-II) was determined by real-time PCR, demonstrating an inducible KLF5 expression in pancreatic cancer cells (*, P < 0.01). C. The effect of IL-1β (10 ng/mL) on cancer cell migration (HPAF-II) was determined in modified Boyden chambers after 48 h (*, P < 0.01). IL-1β increased pancreatic cancer cell motility. D. Treatment of BxPC3 cells with a function-blocking anti–IL-1R antibody (10 μg/mL, 72 h) diminished constitutive KLF5 mRNA expression as determined by real-time PCR (*, P < 0.05). E. Signaling inhibitors to Erk1/2 (UO126), PI3K/Akt (LY), SAPK (SP), and p38 (SB) were used to identify pathways involved in IL-1β–mediated KLF5 up-regulation. Changes in KLF5 expression (HPAF-II) were determined after 24 h by real-time PCR (*, P < 0.01, versus control + IL-1β). Experiments were done in triplicate. Columns, mean; bars, SEM.

Article Snippet: The anti-KLF5 antibody was obtained from R&D Systems.

Techniques: Expressing, Western Blot, Protein-Protein interactions, Real-time Polymerase Chain Reaction, Migration, Modification, Blocking Assay, Control

Journal: Molecular Cancer Research

Article Title: Up-Regulation of Krüppel-Like Factor 5 in Pancreatic Cancer Is Promoted by Interleukin-1β Signaling and Hypoxia-Inducible Factor-1α

doi: 10.1158/1541-7786.mcr-08-0525

Figure Lengend Snippet: FIGURE 3. Role of HIF-1α in KLF5 regulation in pancreatic cancer. A. Western blot analysis for HIF-1α protein was done to determine the effects of IL-1β. Results show a time-dependent stimulation of cancer cells with IL-1β (10 ng/mL) and a consecutive increase in nuclear HIF-1α protein (HPAF-II). B. The effect of hypoxia on KLF5 expression was investigated. Cells were incubated under either hypoxic (1% O2) or nonhypoxic conditions (20% O2), and real-time PCR was done thereafter to determine the effect on KLF5 mRNA expression (*, P < 0.05). C. Similarly, chemical hypoxia with desferroxamine (DFX; 100 μmol/L) time-dependently up-regulated KLF5 protein levels in BxPC3 cells as determined by Western blotting. D. A potential interaction of KLF5 and HIF-1α was investigated by immunoprecipitation (IP) and Western blotting. Results show that KLF5 coimmunoprecipitates with HIF-1α (IP: HIF-1α), independent of stimulation. E. HIF-1α protein coimmunoprecipitated with KLF5 (IP: KLF5) and stimulation with IL-1β increased the amount of HIF-1α that complexed with KLF5. F. Effect of HIF-1α down-regulation on KLF5 expression. Transient RNAi of HIF-1α (stealth siRNA) was done. Blocking HIF-1α lowered KLF5 expres- sion (48 h) under constitutive conditions. G. Similarly, inhibition of KLF5 by transient RNAi (48 h) diminished the expression of the HIF-1α target gene GLUT-1 in BxPC3 pancreatic cancer cells (*, P < 0.05). Columns, mean; bars, SEM.

Article Snippet: The anti-KLF5 antibody was obtained from R&D Systems.

Techniques: Western Blot, Expressing, Incubation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Blocking Assay, Inhibition

Journal: Molecular Cancer Research

Article Title: Up-Regulation of Krüppel-Like Factor 5 in Pancreatic Cancer Is Promoted by Interleukin-1β Signaling and Hypoxia-Inducible Factor-1α

doi: 10.1158/1541-7786.mcr-08-0525

Figure Lengend Snippet: FIGURE 4. Effect of cell density on KLF5 expression. A. Effects of cell density on KLF5 expression. Cells (HPAF-II) were grown to the indicated monolayer cell confluence and real-time PCR for KLF5 mRNA expression was done (*, P < 0.01; n = 3/group). B. Viable anchorage-independently growing cells were collected from culture medium supernatants and the remaining adherent cells were isolated for RNA extraction to determine KLF5 mRNA by PCR. Anchorage-independently growing cells elicited a significantly higher KLF5 mRNA expression (*, P < 0.01). C. Pancreatic cancer cells (HPAF-II and L3.6pl) were grown either in monolayer (M; con- fluence) or as tumor cell spheroids (S) over 3 d. Changes in KLF5 mRNA expression were evaluated by real-time PCR (*, P < 0.05). Columns, mean; bars, SEM.

Article Snippet: The anti-KLF5 antibody was obtained from R&D Systems.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, RNA Extraction

Journal: Molecular Cancer Research

Article Title: Up-Regulation of Krüppel-Like Factor 5 in Pancreatic Cancer Is Promoted by Interleukin-1β Signaling and Hypoxia-Inducible Factor-1α

doi: 10.1158/1541-7786.mcr-08-0525

Figure Lengend Snippet: FIGURE 5. Effects of KLF5 knockdown on pancreatic cancer cells. A. KLF5 siRNA (stealth siRNA) was transiently transfected into BxPC3 pan- creatic cancer cells. Both siRNA constructs markedly diminished KLF5 protein after 48 h as determined by Western blotting. B. Analysis of PDGF-A mRNA expression. Transient KLF5 inhibition by RNAi (48 h) in BxPC3 cells led to a significant reduction in PDGF-A expression (*, P < 0.01). C. Similarly, survivin mRNA expression was markedly reduced upon transient KLF5 inhibition (48 h; BxPC3 cells; *, P < 0.01). Columns, mean; bars, SEM.

Article Snippet: The anti-KLF5 antibody was obtained from R&D Systems.

Techniques: Knockdown, Transfection, Construct, Western Blot, Expressing, Inhibition

Journal: Theranostics

Article Title: TGF-β causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

doi: 10.7150/thno.44567

Figure Lengend Snippet: Figure 1. KLF5 is required by TGF-β to induce DTX resistance in prostate cancer cells. (A, B) Cytotoxicity assay in prostate cancer DU 145 and PC-3 cells with concomitant treatment with docetaxel (DTX) and TGF-β1 (10 ng/µl) and/or TGF-β receptor I inhibitor, SB505124 (SB, 2.5 µM). (C, D) Cytotoxicity assay in DU 145 and PC-3 cell variants with concomitant treatment with DTX and TGF-β1 (10 ng/µl) and/or SB505124 (2.5 µM). KLF5 -/-, endogenous KLF5 knockout. (E, F) Colony formation assay of KLF5 -/- DU 145 and PC-3 cells with or without wild type KLF5 restoration in Matrigel treated with DTX (1 nM) and/or TGF-β1 (10 ng/µl). Cytotoxicity assay and Matrigel colony formation assay were performed in triplicate, and error bars represent the standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. DTX: docetaxel; SB: SB-505124.

Article Snippet: Cell lysate, the supernatant, was incubated with HA-Tag antibody (3724; Cell Signaling Technology) or KLF5 antibody (AF 3758; R&D Systems) overnight at 4 °C.

Techniques: Cytotoxicity Assay, Knock-Out, Colony Assay

Journal: Theranostics

Article Title: TGF-β causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

doi: 10.7150/thno.44567

Figure Lengend Snippet: Figure 2. KLF5 acetylation at K369 mediates DTX resistance in prostate cancer cells. (A) Cytotoxicity assay in DU 145 (KLF5-/-) cells expressing wild type KLF5 and acetylation deficient mutant KLF5K369R (KR) with concomitant treatment with DTX and TGF-β1 (10 ng/µl) and/or SB505124 (2.5 µM). (B) Cytotoxicity assay (left) and colony formation assay in Matrigel (right) of DU 145 (KLF5-/-) cells expressing acetylation deficient mutant KLF5K369R (KR) and acetylation mimicking mutant KLF5K369Q (KQ) treated with DTX (1 nM). (C - F) Cytotoxicity assay of DTX (C, D) and colony formation assay with 1 nM DTX (E, F) in DU 145 and PC-3 (KLF5-/-) cells expressing KR and KQ with concomitant treatment of TGF-β1 (10 ng/µl) and/or SB505124 (2.5 µM). (G - I) Xenograft tumorigenesis assay with DU 145 KLF5 -/- (EV), wild-type KLF5 (KLF5), KR, KQ cells. Eight tumors from 4 nude mice were available for each group. Cytotoxicity assay and Matrigel colony formation assay were performed in triplicate, and error bars represent the standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. DTX: docetaxel; SB: SB-505124.

Article Snippet: Cell lysate, the supernatant, was incubated with HA-Tag antibody (3724; Cell Signaling Technology) or KLF5 antibody (AF 3758; R&D Systems) overnight at 4 °C.

Techniques: Cytotoxicity Assay, Expressing, Mutagenesis, Colony Assay

Journal: Theranostics

Article Title: TGF-β causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

doi: 10.7150/thno.44567

Figure Lengend Snippet: Figure 3. Acetylated KLF5 induces DTX resistance by upregulating Bcl-2. (A) Western blotting analysis of Bcl-2 family proteins in isogenic KLF5 null (KLF5 -/-) DU 145 cells expressing empty vector (EV), KLF5WT (KLF5), KLF5K369R (KR), and KLF5K369Q (KQ). β-actin is used as endogenous control. (B) Mapping of promoter of BCL2 mRNA regulated by acetylated KLF5 by transfecting BCL2 promoter truncations with pGL3 plasmid backbone in DU 145 EV, KLF5, KR, KQ cells. (C) Relative luciferase activities in EV, KQ and KQ cells transfected with KLF5 siRNA. (D) Relative mRNA levels of BCL2 in DU 145 EV, KLF5, KR, and KQ cells, as detected by real-time qPCR with GAPDH as endogenous control. (E) Detection of Bcl-2 and β-actin (endogenous control) proteins by Western blotting after KLF5 knockdown by siRNA in DU 145 KR and KQ cells. (F, G) Cytotoxicity assay of DTX in DU 145 KQ and KR cells treated with Bcl-2 inhibitor, ABT-199 (500 nM). Real time qPCR assay, and cytotoxicity assay were performed in triplicate, and error bars represent the standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. DTX: docetaxel.

Article Snippet: Cell lysate, the supernatant, was incubated with HA-Tag antibody (3724; Cell Signaling Technology) or KLF5 antibody (AF 3758; R&D Systems) overnight at 4 °C.

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Luciferase, Transfection, Knockdown, Cytotoxicity Assay

Journal: Theranostics

Article Title: TGF-β causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

doi: 10.7150/thno.44567

Figure Lengend Snippet: Figure 4. TGF-β induces KLF5 acetylation and upregulates Bcl-2 expression. (A) Western blotting analyses of KLF5 and Bcl-2 in whole cell protein lysates and acetylated lysine in KLF5 immunoprecipitated protein lysates, after 0, 24, 48, 72 hours treatment with 10 ng/ml TGF-β1. (B) Relative luciferase activities in KLF5 cells transfected with BCL2 promoter and treated with TGF-β1. (C, D) Relative mRNA levels of BCL2 in DU 145 EV, KLF5, KR, and KQ cells treated with 0, 0.2, 2, 10 ng/µl TGF-β1 for 48 hours (C), TGF-β1 (10 ng/µl) for 0, 24, 48, 72 hours (D), as detected by real-time qPCR with GAPDH as endogenous control. (E) Relative mRNA level of BCL2 in DU 145 KQ cells treated with 2.5 nM SB-505124 for 0, 24, 48, 72 hours, as detected by real-time qPCR with GAPDH as endogenous control. (F) Relative luciferase activities in KQ cells transfected with BCL2 promoter and treated with SB-505124 (SB, 2.5 µM). Real-time qPCR assay, and luciferase activity assay were performed in triplicate, and error bars represent the standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Cell lysate, the supernatant, was incubated with HA-Tag antibody (3724; Cell Signaling Technology) or KLF5 antibody (AF 3758; R&D Systems) overnight at 4 °C.

Techniques: Expressing, Western Blot, Immunoprecipitation, Luciferase, Transfection, Control, Activity Assay

Journal: Theranostics

Article Title: TGF-β causes Docetaxel resistance in Prostate Cancer via the induction of Bcl-2 by acetylated KLF5 and Protein Stabilization.

doi: 10.7150/thno.44567

Figure Lengend Snippet: Figure 6. Higher acetylated KLF5 correlates with higher Bcl-2 expression, and the latter associates with poorer survival of prostate cancer patients. (A) Kaplan-Meier estimates of disease free survival in 247 advanced stage prostate cancer patients with KLF5 mRNA level greater than median (TCGA, Provisional). (B) mRNA expression analysis and correlation of BCL2 and KLF5 from 499 prostate cancer patient samples (TCGA, Provisional). (C) Kaplan-Meier estimates of disease-free survival in 267 patients with prostate cancer (TCGA, Provisional). Bcl-2low: protein expression z-score less than mean. Bcl-2high: protein expression z-score greater than mean. (D) BCL2 mRNA expression level in taxane treated prostate cancer patients with high and low prostate specific antigen (PSA) level. (E) Prostate cancer tissue arrays stained for acetylated KLF5 and Bcl-2 pictured to show correlation of acetylated KLF5 and Bcl-2. Two representative tumor samples are shown. (F) A schematic model shows that TGF-β and acetylated KLF5 signaling axis induce DTX resistance through Bcl-2 upregulation and stabilization. Error bars represent the standard errors of the means. ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. Scale bars, 100 µm. Magnification, X40. DTX: docetaxel.

Article Snippet: Cell lysate, the supernatant, was incubated with HA-Tag antibody (3724; Cell Signaling Technology) or KLF5 antibody (AF 3758; R&D Systems) overnight at 4 °C.

Techniques: Expressing, Staining

Journal: Non-coding RNA Research

Article Title: MicroRNA miR-214-5p induces senescence of microvascular endothelial cells by targeting the JAG1/Notch signaling pathway

doi: 10.1016/j.ncrna.2023.05.002

Figure Lengend Snippet: Identification of miR-214-5p target gene. (A) The expression of candidate target genes of miR-214-5p was analyzed by qRT-PCR in serially passaged MVECs. (B) KLF5 and JAG1 protein levels were determined via immunoblotting analysis in serially passaged MVECs. (C) KLF5 and JAG1 protein levels were determined in young MVECs treated with miR-214-5p mimic and old MVECs treated with miR-214-5p inhibitor. (D) Luciferase activities were measured after co-transfection with the luciferase reporter gene and a miR-214-5p mimic into 293T cells. The luciferase reporter gene was fused with 3′-UTR sequences of a wild-type JAG1 (wt) or a mutated sequence (mut).

Article Snippet: Membranes were immunoblotted with antibodies against KLF5 (Novus, Littleton, CO, USA), Hairy/Enhancer-of-split related with YRPW motif protein 1 (HEY1; Abcam, Cambridge, UK), Hairy/Enhancer of Split-1 (HES1; Cell Signaling, Berkeley, CA, USA), and JAG1 and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), and detected via chemiluminescence using enhanced chemiluminescence (ECL) detection reagents.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Luciferase, Cotransfection, Sequencing