Journal: Biochemistry and Biophysics Reports

Article Title: Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

doi: 10.1016/j.bbrep.2020.100841

Figure Lengend Snippet: Study scheme: conceptual feature (A) and experimental workflow (B) of this research. (A) Conceptual feature of this research was to identify the difference between the conventional method (Y-27632 is contained in the medium and cells) and the proposed novel method “pretreatment” (Y-27632 is not contained in the medium but in cells). (B) Experimental workflow of this research. RPE cells were cultured for 2 days as adhesion (transient exposure was performed 2 h before its end) and then suspended under one of the three conditions (PC, NC, and pretreatment). After suspension culture, cells were utilized for each analysis in this study.

Article Snippet: Human primary RPE cells (#00194987, Lonza) were cultured in a maintenance medium (DMEM/F12 [7:3]) supplemented with B27 (Thermo Fisher Scientific) and 2 mM l -glutamine (FUJIFILM Wako Pure Chemical Corporation) and 10 ng/mL bFGF and 0.5 μM SB43152 on Laminin-511 E8 fragment (iMatrix-511, Nippi)-coated plates in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Cell Culture, Suspension

Journal: Biochemistry and Biophysics Reports

Article Title: Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

doi: 10.1016/j.bbrep.2020.100841

Figure Lengend Snippet: The number of viable human RPE cells after suspension culture in each condition: negative control (NC), positive control (PC), and pretreatment condition. (A) Evaluation of the number of viable cells after 4 h of suspension culture in each condition. The Y-27632 concentrations used were 1, 5, 10, 30, and 100 μM. (B) Evaluation of the number of viable cells after suspension culture with 10 μM Y-27632 for 4 and 8 h. (A, B) n = 5; mean ± SED; *p < 0.05 and **p < 0.01 compared with all other conditions; one-way ANOVA with Tukey's post hoc pairwise comparisons test.

Article Snippet: Human primary RPE cells (#00194987, Lonza) were cultured in a maintenance medium (DMEM/F12 [7:3]) supplemented with B27 (Thermo Fisher Scientific) and 2 mM l -glutamine (FUJIFILM Wako Pure Chemical Corporation) and 10 ng/mL bFGF and 0.5 μM SB43152 on Laminin-511 E8 fragment (iMatrix-511, Nippi)-coated plates in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Suspension, Negative Control, Positive Control

Journal: Biochemistry and Biophysics Reports

Article Title: Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

doi: 10.1016/j.bbrep.2020.100841

Figure Lengend Snippet: Functional assessment and cell signature in recovery culture. (A) Cell proliferation assessment by MTS assay: RPE cells after 4 h suspension culture in NC, PC, and pretreatment conditions were plated for recovery culture, and cell proliferation was subsequently evaluated by MTS assay each day from days 1–4 (n = 3). (B) Epithelial morphology and then number of viable cells at confluence during recovery culture at 10 days after suspension culture. Cells were plated into a 12-well laminin-coated plate and cultured for 10 days using RPE cells after suspension culture. (B-1) Morphology was assessed by immunofluorescence microscopy (green, ZO-1). Upper is low magnification (10 × ) and lower is high magnification (20 × ). Scale bars = 100 μm. (B-2) The number of viable cells in the image of samples prepared for (B-1) was quantified (n = 3; mean ± SED; *p < 0.05 and **p < 0.01). (C) VEGF secretion quantified using an ELISA assay in preserved RPE cells after 13 days in a recovery culture (n = 4). NC, negative control; PC, positive control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Human primary RPE cells (#00194987, Lonza) were cultured in a maintenance medium (DMEM/F12 [7:3]) supplemented with B27 (Thermo Fisher Scientific) and 2 mM l -glutamine (FUJIFILM Wako Pure Chemical Corporation) and 10 ng/mL bFGF and 0.5 μM SB43152 on Laminin-511 E8 fragment (iMatrix-511, Nippi)-coated plates in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Functional Assay, MTS Assay, Suspension, Cell Culture, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: Biochemistry and Biophysics Reports

Article Title: Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

doi: 10.1016/j.bbrep.2020.100841

Figure Lengend Snippet: mRNA expression analysis of RPE cells after transient exposure of suspension culture to Y-27632. (A) Schematic of relationship between Y-27632 and cell death. RPE cell dissociation triggers apoptotic mechanisms via RhoA/ROCK signaling pathway that progress to cell death and Y-27632 involvement. (B) mRNA expression level of RhoA and ROCK2 in human RPE cells pretreated with 10 μM Y-27632 after 4 h suspension culture (ΔΔCt to β-actin ). Data are shown as mean ± SED (n = 4). One-way ANOVA with Tukey's post hoc pairwise comparison tests were performed. NC, negative control; PC, positive control.

Article Snippet: Human primary RPE cells (#00194987, Lonza) were cultured in a maintenance medium (DMEM/F12 [7:3]) supplemented with B27 (Thermo Fisher Scientific) and 2 mM l -glutamine (FUJIFILM Wako Pure Chemical Corporation) and 10 ng/mL bFGF and 0.5 μM SB43152 on Laminin-511 E8 fragment (iMatrix-511, Nippi)-coated plates in a humidified incubator at 37 °C in 5% CO 2 .

Techniques: Expressing, Suspension, Comparison, Negative Control, Positive Control

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: A, All sequence were cloned into yeast p416 GPD expression vector containing a URA3 marker and GPD promoter for constitutive expression. Yeast strain BY4741 was transformed with the indicated constructs, and nuclei were stained with Hochest. The localization of GFP fusion proteins and Hochest staining were visualized by fluorescence microscopy. B, Tetrad analysis of mutant 72.5E. Mutant 72.5E was crossed to wild-type strain BY4741. The diploids were then sporulated, and the resultant haploid spores were separated by microdissection and grown at 30°C for 3 days. Resultant spores showed 2:2 segregation of colony size and GFP nuclear localization. Small colony phenotype segregated with nuclear GFP localization. C, Rescue screen to identify mutated gene. Mutant yeast cells were transformed with wild-type library. Individually transformed cells grown at permissive temperature (30°C, left plate) or restrictive temperature (39°C, right plate). D, The localizations of GFP fusion protein were only rescued by the transformation of wild-type Prp8 vector, but not by wild-type Prp43 or control vector, shown by fluorescence microscopy.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Sequencing, Clone Assay, Expressing, Plasmid Preparation, Marker, Transformation Assay, Construct, Staining, Fluorescence, Microscopy, Mutagenesis, Laser Capture Microdissection

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: Prp8 knockdown inhibited NESAR nuclear export in rapamycin assay. Prp8 was stably knockdown in PC3 cells by shRNA (C and D), and control cells were transfected with scramble shRNA (A and B). Cells were cotransfected with RFP-NLS-FKBP and GFP-FRB (A and C), or cells were cotransfected with RFP-NLS-FKBP and GFP-NESAR-FRB (B and D). Transfected cells were treated with EtOH (control) or rapamycin at 0.5μM for 30 minutes (Ligand). Ligand was withdrawn (W/d) 30 minutes after rapamycin treatment, and cells were cultured for an additional 2 or 18 hours. Cycloheximide (CHX) at 50 μg/mL was added to inhibit protein synthesis in18-hour W/d experiment. The localization of GFP and RFP fusion proteins was observed by fluorescent microscopy. The % of transfected cells exhibiting predominant nuclear localization of GFP-FRB or GFP-NESAR-FRB is indicated. The experiment was reproduced 3 times. E, Localization of GFP-AR in PC3 cells transfected with control siRNA or 2 different siRNAs targeting Prp8. F, Quantitative result shows the percentage of cells with evenly distributed GFP-AR in the cytoplasm and nuclei of PC3 cells.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Stable Transfection, shRNA, Transfection, Cell Culture, Microscopy

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: Prp8 expression is decreased in castration-resistant LuCaP35 xenograft tumors and advanced PCa specimens. Prp8 (A), PSA (B), and AR (C) expression levels in LuCaP35 xenograft tumors were examined by quantitative real-time PCR. D, Prp8 expression levels in LuCaP35 xenograft tumors were examined by Western blottings. A-S, androgen-sensitive tumors showed response to castration by decreased volume; C-R, castration-resistant tumors continued growing after castration and were further transplanted to new castrated mice. E, Human prostate tissues with Gleason score 7+ were acquired (n = 14). Epithelial cells were laser-capture microdissected and used for real-time PCR analysis. Prp8 mRNA levels were quantified and normalized to GAPDH. All data represent matched pairs of cancer (C) and normal adjacent tissue (NA). Wilcoxian rank sum test was performed. *, P < .05.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: Prp8 physically interacts with AR in prostate cancer cells. A, LNCaP cells were cotransfected with Flag-PRP8 and GFP or GFP-AR. Immunoprecipitation was performed with agarose beads conjugated with anti-GFP antibody. Western blottings were performed with anti-Flag antibody. B, LNCaP cell lysates were incubated with anti-AR antibody or normal rabbit IgG overnight followed with incubation with agarose beads for 3 hours. C, LNCaP cells were cotransfected with Flag-Prp8 with GFP, GFP-NAR, or GFP-LBD, respectively. Immunoprecipitation was performed with agarose beads conjugated with anti-GFP antibody. Western blottings were performed with anti-Flag antibody. D, LNCaP cells were cotransfected with Flag-Prp8 with GFP, GFP-NES, or GFP-AR(ΔNES). Immunoprecipitation was performed with agarose beads conjugated with anti-GFP antibody. Western blottings were performed with anti-Flag antibody.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Immunoprecipitation, Western Blot, Incubation

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: Prp8 regulates AR transactivation activity. A and B, Prp8 and PSA expression levels were examined by Western blotting of LNCaP cells with a stable knockdown of Prp8. GAPDH served as loading control. C and D, Luciferase reporter assay using the 6.1-kb PSA promoter-driven luciferase reporter in Prp8 knockdown and scramble control LNCaP cells (C) and in Prp8-overexpressing and control LNCaP cells (D). The Renilla luciferase gene was used as an internal control in the luciferase assay. **, P < .01. Experiments were repeated 3 times.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Activity Assay, Expressing, Western Blot, Luciferase, Reporter Assay

Journal: Molecular Endocrinology

Article Title: Splicing Factor Prp8 Interacts With NES AR and Regulates Androgen Receptor in Prostate Cancer Cells

doi: 10.1210/me.2015-1112

Figure Lengend Snippet: Knockdown Prp8 increases AR ubiquitination. Prp8 expression was knocked down in LNCaP (A) and C4–2 (B) cells by siRNA transfection. Cell lysates were incubated with anti-AR antibody overnight followed with incubation with agarose beads for 3 hours. Ubiquitin, AR, and Prp8 expression levels were examined by Western blotting. Experiments were repeated 3 times.

Article Snippet: A plasmid vector containing human Prp8 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_006445","term_id":"91208425","term_text":"NM_006445"}} NM_006445 ) was obtained from Origene (sc116070), and it was used as a PCR template to construct Prp8 expression vectors with different epitope-tags by routine molecular cloning techniques.

Techniques: Expressing, Transfection, Incubation, Western Blot

Journal: Pharmaceutics

Article Title: Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties

doi: 10.3390/pharmaceutics11080412

Figure Lengend Snippet: LEPI cells form similar tight junctions and microvilli with primary RPE cells. SEM imaging was used to evaluate the microvilli formation ( a , c , e ). ARPE19 cells ( a ) do not form clear microvilli, whereas hfRPE cells ( c ) and LEPI cells ( e ) display microvilli. Scale bars of SEM images ( a , c , e ) are 200 nm. The TEM images show tight junctions (circled) in ARPE19 ( b ), hfRPE ( d ) and LEPI cells ( f ). ARPE19 TEM image scale bar is 2 µm ( b ), and the LEPI ( f ) and hfRPE ( d ) image scale bars are 500 nm.

Article Snippet: Human primary RPE cells (hfRPE cells, HRPEpiC, ScienCell, Carlsbad, CA, USA) were expanded and cultured, as described previously [ ].

Techniques: Imaging

Journal: Pharmaceutics

Article Title: Retinal Pigment Epithelial Cell Line with Fast Differentiation and Improved Barrier Properties

doi: 10.3390/pharmaceutics11080412

Figure Lengend Snippet: LEPI cells with mature RPE phenotype. Occludin was used in all ( a – c ) images to reveal the cell shape and the presence of tight junctions (green). DAPI stain was used to visualize the cell nuclei (blue). ( a ) Ezrin (red) was detected on the apical surface indicating proper microvilli formation. ( b ) LEPI cells showed RPE-specific BEST1 expression on the basal membrane (red) and ( c ) RPE65 expression in the cytoplasm (red). The cells were cultured 18 days before imaging. ( d ) Confocal microscope image of LEPI cells cultured on Ibidi slides for one week. The cells were exposed to FITC labeled photoreceptor outer segments (FITC-POS, green), and after 20 h incubation, the cells were washed and counterstained with anti-MRP1 antibody (red) localized in lateral plasma membranes. The vertical section view (upper panel) shows internalized FITC-POS in LEPI cells. Scale bars 10 µm.

Article Snippet: Human primary RPE cells (hfRPE cells, HRPEpiC, ScienCell, Carlsbad, CA, USA) were expanded and cultured, as described previously [ ].

Techniques: Staining, Expressing, Membrane, Cell Culture, Imaging, Microscopy, Labeling, Incubation, Clinical Proteomics