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: The Journal of Biological Chemistry

Article Title: Transducin-like Enhancer of Split-1 (TLE1) Combines with Forkhead Box Protein G1 (FoxG1) to Promote Neuronal Survival *

doi: 10.1074/jbc.M111.328336

Figure Lengend Snippet: Effects of TLE1 knockdown in CGNs. A, Western blot analysis of endogenous TLE1 protein after knockdown using sh1 and sh2 shRNA in HEK 293T cells. sh1 was able to completely knock down endogenous levels of TLE1, whereas sh2 had a modest effect. Shown is densitometric analysis of endogenous TLE1 protein levels from three independent experiments after knockdown using sh1 and sh2 shRNA. B, viability of CGNs transfected with shRNAs, sh1 and sh2, against TLE1. Knockdown of endogenous TLE1 kills healthy neurons treated with HK. Viability was normalized with CGNs transfected with control shRNA treated with HK. Error bars, S.E. **, p <0.01; ***, p < 0.001.

Article Snippet: Two shRNA constructs against TLE1 and a control shRNA construct were purchased from Origene.

Techniques: Knockdown, Western Blot, shRNA, Transfection, Control

Journal: eLife

Article Title: Multiple cullin-associated E3 ligases regulate cyclin D1 protein stability

doi: 10.7554/elife.80327

Figure Lengend Snippet: Figure 1. Cullin-associated E3 ligases mediate cyclin D1 ubiquitination and proteasome degradation. (A) Co-immunoprecipitation (co-IP) of Keap1, CUL3 with endogenous cyclin D1. HA-Keap1 and Myc-CUL3 were co-transfected into HEK293 cells with the MG132 treatment (10 μM, 4 hr incubation). 24 hr after transfection, the cell lysates were collected. To detect interaction of Keap1 with cyclin D1 or CUL3, co-IP was performed using the anti-HA (α-HA) antibody followed by the western blot using the anti-cyclin D1 or anti-Myc (α-Myc) antibody. To detect the interaction between CUL3 with cyclin D1, co-IP assay was performed using the anti-Myc antibody followed by the western blot using the anti-cyclin D1 antibody. (B) Co-IP of DDB2 and CUL4A/4B with endogenous cyclin D1. Myc-DDB2, HA-CUL4A, and Flag-CUL4B were transfected into HEK293 cells with the MG132 treatment (10 μM, 4 hr incubation). Co-IP was performed using the anti-Flag (α-Flag), anti-HA, or anti-Myc antibody followed by the western blot using the anti-cyclin D1 antibody. (C) Co-IP of WSB2 and CUL2/5 with endogenous cyclin D1. Wild-type (WT) or mutant form of WSB2 (SOCS∆364–400) were co-transfected with Myc-CUL2/5 into HEK293 cells with MG132 treatment (10 μM, 4 hr incubation). Co-IP was performed using the anti-Myc or anti-WSB2 (α-WSB2) antibodies followed by the western blot using the anti-cyclin D1 antibody. (D) Co-IP of Rbx1 with endogenous cyclin D1. Myc-Rbx1 was transfected into HEK293 cells with MG132 treatment (10 μM, 4 hr incubation). Co-IP assay was performed using the anti-WSB2 or anti-Myc antibody followed by the western blot using the anti-cyclin D1 antibody. (E–H) Ubiquitination assay. WT or mutant cyclin D1 (T286A) were co-transfected with WT or mutant Keap1, DDB2, WSB2, or Rbx1 expression plasmids into HEK293 cells with the treatment of MG132 (10 μM, 4 hr incubation). Co-IP was performed using the anti-Ub (α-Ub) antibody followed by the western blot using the anti-cyclin D1 (α-cyclin D1) antibody. (I–L) WT or mutant Keap1, DDB2, WSB2, and Rbx1 expression plasmids were co-transfected with cyclin D1 expression plasmid into HEK293 cells with the MG132 treatment (10 μM, 4 hr incubation). Cyclin D1 protein levels were detected by the western blot analysis. (M, N) Protein decay assay. HEK293 cells were transfected with scramble siRNA (Ctrl) or Keap1, DDB2, WSB2, or Rbx1 siRNA. The cell lysates were collected 0, 30, 60, 120, or 300 min after cycloheximide treatment (80 μg/ml) and the cyclin D1 protein levels were detected by the western blot analysis and were quantified (n=3).

Article Snippet: The plasmid pCMV6- WSB2 (NM_018639.3) was purchased from OriGene.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Incubation, Western Blot, Mutagenesis, Expressing, Plasmid Preparation

Journal: eLife

Article Title: Multiple cullin-associated E3 ligases regulate cyclin D1 protein stability

doi: 10.7554/elife.80327

Figure Lengend Snippet: Figure 2. Cullin-associated E3 ligases affect cyclin D1 function. (A, D, G, J) Keap1, DDB2, WSB2, or Rbx1 siRNAs were transiently transfected into human colon cancer cell line HCT-116 cells. Endogenous cyclin D1 protein levels were detected by the western blot using the anti-cyclin D1 antibody. (B, E, H, K) Cell proliferation assay. Keap1, DDB2, WSB2, or Rbx1 expression plasmids were transfected into HCT-116 cells which were stably transfected with wild-type (WT) or mutant cyclin D1 (T286). The cells were stained with crystal violet 5 days after Keap1, DDB2, WSB2, or Rbx1 transfection. (C, F, I, L) Bromodeoxyuridine (BrdU) incorporation assay. Keap1, DDB2, WSB2, or Rbx1 expression plasmid was transiently transfected into HCT-116 cells which were stably transfected with WT or mutant cyclin D1 (T286). 4 hr before the harvest of cells, the cells were treated with BrdU (20 μM). 48 hr after the transfection, BrdU incorporation assays were performed (n=3). Data were presented as means ± SD of three independent experiments. Statistical analyses were performed using two-way ANOVA followed by the Tucky’s post-hoc test, *P<0.05 (n=3). (M) A model showing that cullin-associated ubiquitin ligases are participated in the cyclin D1 proteolysis process.

Article Snippet: The plasmid pCMV6- WSB2 (NM_018639.3) was purchased from OriGene.

Techniques: Transfection, Western Blot, Proliferation Assay, Expressing, Stable Transfection, Mutagenesis, Staining, BrdU Incorporation Assay, Plasmid Preparation, Ubiquitin Proteomics

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: (A) Experimental strategy to record mEPSCs in mouse primary HP neuronal cultures following KD of KIF5C and KIF3A using shRNAs. (B) eGFP-labeled neurons in fluorescence and bright-field (BF) images with patch-clamp recording electrodes attached. (C) Two traces of mEPSCs for negative control, or NC (scrambled shRNA), and shRNAs against KIF5C and KIF3A . (D and E) Changes in amplitudes and frequencies of mEPSCs 72 h after KD of KIF5C and KIF3A . Number of neurons patched per group is labeled. Data (mean) from all KD groups compared to negative control. (F and G) Changes in amplitude and frequency, respectively, of mEPSCs between NC and KIF5C or KIF3A shRNA (Kolmogorov-Smirnov test, p < 0.05). (H) Experimental plan for KIF5C KD and confocal live imaging. (I and J) Confocal projection images showing changes in spine, digitally enlarged image in inset. (K–N) Quantitative analyses of image data shown in (I) and (J) executed by selecting dendrites with length of 100 μm. Bar graphs show change in spine density or morphology. (O and P) Confocal projection images show soma in center to depict dendritic arbor. (Q and R) Quantification of dendritic morphology changes in terms of number of intersections per 20-μm step size by Sholl analysis. Changes compared between KIF5C KD or KIF3A KD and NC. One-way ANOVA followed by Tukey’s post hoc test. Error bars represent SEM. *p < 0.05, *p < 0.005, and ***p < 0.0005. Scale bar: 2 or 20 μm.

Article Snippet: pEGFP-C-shLenti against KIF5C (catalog# TL511686), pEGFP-C-shLenti against KIF3A (catalog# TL516184) and a scrambled shRNA control (catalog# TR30013) were obtained from ORIGENE. pEGFP- KIF5C -N1 was prepared at Puthanveettil Lab and pGL-FL KIF3A was a gift from Linda Wordeman (Addgene plasmid # 13742).

Techniques: Labeling, Fluorescence, Patch Clamp, Negative Control, shRNA, Imaging

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: (A) Experimental strategy to record mEPSCs in mouse primary HP neuronal cultures following OE of KIF5C and KIF3A using full-length cDNA expressing plasmids. (B) Two traces of mEPSCs for control, KIF5C OE and KIF3A OE. (C and D) Changes in amplitudes and frequencies of mEPSCs 24 h after KIF5C OE. (E and F) Changes in amplitude and frequency of mEPSCs between control and KIF5C OE groups (Kolmogorov-Smirnov test, p < 0.05). (G) Experimental plan for overexpressing KIF5C for confocal live imaging. (H) Confocal projection images of KIF5C OE showing spine changes, digitally enlarged image in inset. (I and J) Quantitative analyses of image data shown in (H) executed by selecting dendrites with length of 100 μm. Bar graphs show change in spine density or morphology. (K) Confocal projection images of KIF5C OE show soma in center to depict dendritic arbor. (L) Quantification of dendritic morphology changes in terms of number of intersections per 10-μm step size by Sholl analysis. (M) Experimental plan for KIF3A KD and KIF5C OE in same neuron for confocal live imaging. (N) Confocal projection images showing changes in spine, digitally enlarged image in inset. (O and P) Quantitative analyses of image data shown in (N) achieved by selecting dendrites with length of 100 μm. Bar graphs show change in spine density or morphology. (Q) Confocal projection images show soma in center to depict dendritic arbor. (R) Quantification of dendritic morphology changes in terms of number of intersections per 10-μm step size by Sholl analysis. Changes compared between KIF5C OE and control, or changes compared between neurons with KIF5C OE and KIF3A KD to control eGFP- and RFP-expressing neurons. Unpaired, two-tailed Student’s t test. Error bars represent SEM. *p < 0.05, *p < 0.005, and ***p < 0.0005. Scale bar: 2 or 20 μm.

Article Snippet: pEGFP-C-shLenti against KIF5C (catalog# TL511686), pEGFP-C-shLenti against KIF3A (catalog# TL516184) and a scrambled shRNA control (catalog# TR30013) were obtained from ORIGENE. pEGFP- KIF5C -N1 was prepared at Puthanveettil Lab and pGL-FL KIF3A was a gift from Linda Wordeman (Addgene plasmid # 13742).

Techniques: Expressing, Control, Imaging, Two Tailed Test

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pEGFP-C-shLenti against KIF5C (catalog# TL511686), pEGFP-C-shLenti against KIF3A (catalog# TL516184) and a scrambled shRNA control (catalog# TR30013) were obtained from ORIGENE. pEGFP- KIF5C -N1 was prepared at Puthanveettil Lab and pGL-FL KIF3A was a gift from Linda Wordeman (Addgene plasmid # 13742).

Techniques: Virus, Recombinant, Cell Culture, Saline, Sterility, Live Cell Imaging, Transfection, Protein Extraction, Protease Inhibitor, Labeling, SYBR Green Assay, Ointment, Adhesive, Gel Extraction, Isolation, TA Cloning, Sequencing, Plasmid Preparation, shRNA, Software, Control