pigment epithelial rpe cells  (Eyecyte Inc)

Journal: bioRxiv

Article Title: Differential roles of cyclin-CDK1 complexes in cell migration and invasion

doi: 10.1101/2024.09.24.614768

Figure Lengend Snippet: A. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown retinal pigment epithelial (RPE) cells seeded in CDM migrating over 6 hours, the same cell moving from left to right in each frame shown. B. Average migration speed of RPE cells in CDM across 16 hour timelapse, n = 75 cells per condition analysed across 3 repeats. C. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown RPE cells seeded in CDM transfected with GFP-RFP Raichu- RhoA probe, lifetime of donor GFP channel, colour code lifetime range shown, values in ns: blue denotes shorter lifetime (high activity), green/yellow denotes higher lifetime (low activity). D. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown RPE cells seeded directly into soft collagen and dyed with membrane tension probe FlipperTR, lifetime of 488 excitation, 575-625 emission (colour code lifetime range shown, values in ns: blue/green denote shorter lifetime – lower tension, yellow/red denote longer lifetime – higher tension). E. Average Flipper-TR lifetime of manually drawn rear (blue) and front (red) regions of interest in RPE cells in collagen gels (rear/front regions of the same cell joined by black line), n > 10 cells analysed per condition across 3 repeats. F. Average Flipper-TR lifetime in rear ROI of Control (Ctrl), CyclinA2 (A), CyclinB1 (B) and CyclinA2 + CyclinB1 (A+B) siRNA cells (same RPE cells as in B analysed), n > 10 cells analysed per condition across 3 repeats. Paired student t-tests used in E, one way ANOVA compared to control used in B and F, **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, n.s. denotes p > 0.05 (not significant).

Article Snippet: A2780 human ovarian cancer cells (female) and RT4, J82 and T24 human bladder cancer cells were maintained in RPMI-1640 medium (Sigma-Aldrich) containing L-Glutamine supplemented with 10% (v/v) fetal calf serum, and 1% (v/v) Antibiotic-antimycotic (Sigma-Aldrich); telomerase- immortalized fibroblasts (TIF) cells (used to produce CDMs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) containing L-Glutamine and supplemented with 10% (v/v) fetal calf serum, and 1% (v/v) Antibiotic-antimycotic (Sigma Aldrich); human retinal pigment epithelial cells (RPE) cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) containing L-Glutamine and supplemented with 10% (v/v) fetal calf serum, 1% (v/v) 100x non-essential amino acid solution (Sigma Aldrich).

Techniques: Control, Knockdown, Migration, Transfection, Activity Assay, Membrane

Journal: bioRxiv

Article Title: Differential roles of cyclin-CDK1 complexes in cell migration and invasion

doi: 10.1101/2024.09.24.614768

Figure Lengend Snippet: A. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown retinal pigment epithelial (RPE) cells seeded in CDM migrating over 6 hours, the same cell moving from left to right in each frame shown. B. Average migration speed of RPE cells in CDM across 16 hour timelapse, n = 75 cells per condition analysed across 3 repeats. C. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown RPE cells seeded in CDM transfected with GFP-RFP Raichu- RhoA probe, lifetime of donor GFP channel, colour code lifetime range shown, values in ns: blue denotes shorter lifetime (high activity), green/yellow denotes higher lifetime (low activity). D. Control, cyclinA2, cyclinB1, and cyclinA2 + cyclinB1 concomitant knockdown RPE cells seeded directly into soft collagen and dyed with membrane tension probe FlipperTR, lifetime of 488 excitation, 575-625 emission (colour code lifetime range shown, values in ns: blue/green denote shorter lifetime – lower tension, yellow/red denote longer lifetime – higher tension). E. Average Flipper-TR lifetime of manually drawn rear (blue) and front (red) regions of interest in RPE cells in collagen gels (rear/front regions of the same cell joined by black line), n > 10 cells analysed per condition across 3 repeats. F. Average Flipper-TR lifetime in rear ROI of Control (Ctrl), CyclinA2 (A), CyclinB1 (B) and CyclinA2 + CyclinB1 (A+B) siRNA cells (same RPE cells as in B analysed), n > 10 cells analysed per condition across 3 repeats. Paired student t-tests used in E, one way ANOVA compared to control used in B and F, **** denotes p < 0.0001, *** denotes p < 0.001, ** denotes p < 0.01, n.s. denotes p > 0.05 (not significant).

Article Snippet: A2780 human ovarian cancer cells (female) and RT4, J82 and T24 human bladder cancer cells were maintained in RPMI-1640 medium (Sigma-Aldrich) containing L-Glutamine supplemented with 10% (v/v) fetal calf serum, and 1% (v/v) Antibiotic-antimycotic (Sigma-Aldrich); telomerase- immortalized fibroblasts (TIF) cells (used to produce CDMs) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) containing L-Glutamine and supplemented with 10% (v/v) fetal calf serum, and 1% (v/v) Antibiotic-antimycotic (Sigma Aldrich); human retinal pigment epithelial cells (RPE) cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco) containing L-Glutamine and supplemented with 10% (v/v) fetal calf serum, 1% (v/v) 100x non-essential amino acid solution (Sigma Aldrich).

Techniques: Control, Knockdown, Migration, Transfection, Activity Assay, Membrane

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: CLEM Characterization of Rab8 and Associated Membrane Trafficking Regulators at Primary Cilium Structures

doi: 10.1007/978-1-0716-1346-7_7

Figure Lengend Snippet: CLEM analysis of tRFP-Rab8A and GFP-EHD1 ciliary localization. RPE-1 cells expressing GFP-EHD1 and tRFP-Rab8A. Following fixation cells were imaged by spinning disk confocal imaging and processed for EM. ROI with cells from fluorescence imaging (a) were identified in EM imaging sections at low magnification (b). The same target cilium identified by fluorescence microscopy can be detected by EM at high magnification (c, d). Nuclei are outlined in white dashed line (a, b). Scale bars: 2μm (reproduced from ref. [14] with permission from Springer)

Article Snippet: Human Retinal Pigment Epithelial (RPE-1) cell line immortalized with hTERT from ATCC (ATCC CRL-4000).

Techniques: Expressing, Imaging, Fluorescence, Microscopy

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: CLEM Characterization of Rab8 and Associated Membrane Trafficking Regulators at Primary Cilium Structures

doi: 10.1007/978-1-0716-1346-7_7

Figure Lengend Snippet: CLEM analysis of tRFP-Rab8A and GFP-EHD1 ciliary localization. RPE-1 cells expressing GFP-EHD1 and tRFP-Rab8A. Following fixation cells were imaged by spinning disk confocal imaging and processed for EM. ROI with cells from fluorescence imaging (a) were identified in EM imaging sections at low magnification (b). The same target cilium identified by fluorescence microscopy can be detected by EM at high magnification (c, d). Nuclei are outlined in white dashed line (a, b). Scale bars: 2μm (reproduced from ref. [14] with permission from Springer)

Article Snippet: Cells and Culture Media Human Retinal Pigment Epithelial (RPE-1) cell line immortalized with hTERT from ATCC (ATCC CRL-4000).

Techniques: Expressing, Imaging, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Laminin 111 triggers cell quiescence and long-term survival by inducing IQGAP1-mediated cytosolic scaffolding of ERK and BAD inactivation

doi: 10.1101/2024.08.01.606017

Figure Lengend Snippet: (A) Top, schematic of in vitro cell quiescence protocol: exponentially growing human hTERT-RPE1 cells were plated at low density and grown for 7 days in full serum medium (refreshed every 2 days), at which point they formed homogeneous monolayers. From Day 7, cells were maintained in serum-free medium (refreshed every 2 days), and used on Day 17 for experiments. Middle, representative images of Growing cells (Day2) or Quiescent (Day 17) cells, immunostaining for a plasma membrane marker (anti-CD44, white) and DNA (blue). Bottom, Representative histogram showing the DNA profile of ethanol-fixed growing (blue) and quiescent (red) RPE1 cells stained for DNA with Hoechst33342 and analyzed by flow cytometry. Gates represent the cut-off lines for cell cycle phases G0/G1, S and G2/M, which are quantified in the upper right corner. Percentage of max (% of Max) indicates the number of cells relative to the peak fraction of cells. A minimum of 50,000 cells per condition were analyzed. (B) Mean diameter of growing and quiescent cells in suspension measured with a haemocytometer. N=15 (growing cells) and N=11 (quiescent cells) biological replicates. (C) Protein concentration measurement using Bradford assay. G0 and FACS-sorted G1 cells were lysed in 8M urea, with volumes adjusted to cell numbers. N=4 biological replicates. (D) Left, representative lanes of Coomassie blue-stained SDS- PAGE gel with lysates from G0 and FACS-sorted G1 cells lysed in equal volumes of 8M urea per cell number. Right, color intensity per lane was quantified by densitometry measurement and normalized to G1 cells. N=5 biological replicates. (E) Left, representative immunoblots of total lysates from FACS-sorted G1 and G0 RPE1 cells probed against GAPDH. The upper blot shows loading normalized to cell number and the lower blot shows loading normalized to total protein levels. Right, GAPDH levels per band quantified by densitometry measurement. N=5 (per cell) or N=6 (per μg protein) biological replicates. (F) Left, representative immunoblots of total lysates from G0 and FACS-sorted G1 cells probed against Cyclin D1, PCNA and Ki67. GAPDH served as loading control. Right, Densitometry quantification of 3 independent immunoblots as shown in (Left). (G) Representative fluorescence microcopy images of Growing or G0 cells immunostaining for endogenous (all green) Cyclin D1, Ki67, phosphorylated Ser807/811 Retinoblastoma protein (pRb) and p27 kip1 (p27) and DNA (blue). (H) Mean fluorescence (Log2) form 3 independent experiments of the indicated endogenous immunostainings Cyclin D1, Ki67, pRb and p27 of quiescent cell cultures measured every 2 days over 25 days. (I) Histogram of pRb in G1 and G0 cells analyzed by flow cytometry. After elimination of cell doublets and dead cells, the population with 2n DNA content was used for further analysis. Gates used for pRb quantification are displayed. Percentages of G1 (blue) and G0 (red) cell populations with low and high pRb are listed in the left and right upper corner, respectively. Blank signal is shown in grey. A minimum of 50,000 cells per condition were analyzed. In A and G, the scale bars represent 50 μm. In B, C, D, and F, bars show the mean and error bars represent SEM. P values calculated by Student’s unpaired two-tailed t-tests. *P<0.05, **P<0.001, ***P<0.001, ****P<0.0001

Article Snippet: Exponentially growing Human, normal and diploid, telomerase reverse transcriptase (hTERT)-immortalized retinal pigmented epithelial (RPE-1) cells (ATCC CRL- 4000, called ‘RPE1’ in this study) cells were cultured in ‘Full Medium’ (DMEM:F12 HAM 1:1v/v (Sigma D6421), 0.25% Sodium bicarbonate w/v (Sigma Aldrich S8761), 1mM GlutaMAX-I (Thermo Fisher Scientific A1286001), 1X antibiotic-antimycotic (Thermo Fisher Scientific 15240062), and 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific 11570506).

Techniques: In Vitro, Immunostaining, Membrane, Marker, Staining, Flow Cytometry, Suspension, Protein Concentration, Bradford Assay, SDS Page, Western Blot, Control, Fluorescence, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Twenty-four hours after knockdown, cells were transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assays after mouse A-Myb overexpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assays with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 /M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco's modified Eagle's medium (DMEM; Lonza).

Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification

Journal: Nucleic Acids Research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: A-MYB binds the Cyclin B2 promotor dependent on the cell cycle. RPE-1 cells were cultivated under growth factor withdrawal (starved) und restimulated by growth factor addition to distinguish between a G 0 - and an S/G 2 /M-enriched condition. Promoter binding of DREAM and MMB components was analyzed by ChIP. The relative enrichment of the Cyclin B2 promoter ( A ), the ORC1 promoter (early cell cycle gene) ( D ), and the negative control GAPDHS promoter ( G ) is visualized relative to the input DNA (representative experiment of n = 3). Binding of DREAM components to the Cyclin B2 promoter ( B ), the ORC1 promoter ( E ), and the GAPDHS promoter ( H ) was analyzed relative to the IgG negative control of the starved condition ( n = 3). Binding of the activating MMB components B-MYB and FOXM1 as well as A-MYB to the Cyclin B2 promoter ( C ), the ORC1 promoter ( F ), and the GAPDHS promoter ( I ) was analyzed relative to the IgG negative control of the starved condition (n = 3).). Mean ± SD are given, and significances were calculated by two-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco's modified Eagle's medium (DMEM; Lonza).

Techniques: Binding Assay, Negative Control

Journal: Nucleic Acids Research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: A-MYB compensates for B-MYB loss in proliferation and causes a lower rate of G 2 /M-arrested cells as well as a lower rate of DNA aberrations. siRNA knockdown was performed on HCT116 ( A , B ) and U2OS cells ( C , D ). For flow cytometric analysis of DNA content to evaluate cell cycle distribution, (A) HCT116 ( n = 3) and (C) U2OS ( n = 5) cells were harvested 48 h after knockdown. Significance tests were performed for the change of the cell population ratio between Ctrl KD and the other conditions. To evaluate the change in cell numbers after knockdown, (B) HCT116 ( n = 3) and (D) U2OS cells ( n = 3) were counted every 24 h beginning from the second day after siRNA transfection. To assure a stable knockdown throughout the experiment, cells were re-transfected with siRNA after 72 h. Graphs on the left show exponential regression on a linear scale and graphs on the right display the same regression on a logarithmic scale. ( E ) siRNA knockdown was performed on the transformed cell lines U2OS, HCT116, HEK293 and HeLa, the untransformed cell lines RPE-1, and BJ, as well as the transformed cell line Hep3B with reportedly low A-MYB expression. Cells were counted two days after knockdown. Mean ± SD are given, and significances were calculated by two-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001). Exponential regression curves are displayed with 95% confidence intervals. Significances between exponential regression curves were calculated by Extra sum-of-squares F test (ns, not significant; *** P < 0.001).

Article Snippet: Human fibroblast BJ cells (DSMZ, Braunschweig, Germany), human colorectal carcinoma HCT116 cells (provided by Bert Vogelstein), human embryonic kidney HEK293 cells (DSMZ), human papillomavirus-related cervical adenocarcinoma HeLa cells (DSMZ), human pediatric hepatocellular carcinoma Hep3B cells (DSMZ), human retinal pigment epithelial hTERT immortalized RPE-1 cells (DSMZ), human glioblastoma T98G cells (DSMZ), and human bone osteosarcoma U2OS cells (DSMZ) were cultivated in Dulbecco's modified Eagle's medium (DMEM; Lonza).

Techniques: Knockdown, Transfection, Transformation Assay, Expressing