corneal endothelial cells  (DSMZ)

Journal: Investigative Ophthalmology & Visual Science

Article Title: Iron-Sulfur Clusters and Iron Responsive Element Binding Proteins Mediate Iron Accumulation in Corneal Endothelial Cells in Fuchs Dystrophy

doi: 10.1167/iovs.66.4.23

Figure Lengend Snippet: FECD endothelial tissues show differential expression of proteins necessary for ISC synthesis, mitochondrial fusion, and iron regulation compared to control donor corneal endothelial tissues. (A) ACO1/IREBP1 protein expression is 1.42-fold higher in FECD than in control lysates . (B) ACO2 expression is 1.42-fold lower in FECD than in control tissues; (C) FDX1 expression is 1.57-fold lower in FECD than in control tissues; (D) IREBP2 protein expression is 8.2-fold higher in FECD than in control lysates. (E) MFN2 expression is 1.2-fold lower in FECD than in control tissues; (F) NFS1 expression is twofold higher in FECD than in control tissues; and (G) PCBP1 shows a nonsignificant, 1.2-fold increase in expression in FECD compared to control tissues. Violet-blue dots represent control tissues and magenta dots represent FECD tissues. Error bars correspond to the mean and SEM; * P < 0.05, ** P < 0.01, *** P < 0.001 regarding statistical significance, when compared to the control tissue pool. See for bands.

Article Snippet: Immortalized human corneal endothelial cells (HCEC-B4G12; Leibniz Institute DSMZ, Braunschweig, Germany) were cultured in Opti-MEM I Reduced Serum Medium (Cat. no. 51985091; ThermoFisher) containing the following supplements: 5 ng/mL human epidermal growth factor (Cat. no. E9644; Sigma-Aldrich Corp., St. Louis, MO, USA); 20 ng/mL mouse nerve growth factor 2.5S (Cat. no. N6009; Sigma-Aldrich Corp.); 50 μg/mL gentamicin (Cat. no. IBI02030; IBI Scientific, Dubuque, IA, USA); 100 μg/mL Normocin (Cat. no. ant-nr-1; InvivoGen, San Diego, CA, USA); 0.8 mg/mL chondroitin sulfate (provided by Numedis, Inc, Isanti, MN, USA); 200 mg/L calcium chloride (Cat. no. C7902; Sigma-Aldrich); and 8% fetal bovine serum (Cat. no. S11150; R&D Systems, Flowery Branch, GA, USA).

Techniques: Quantitative Proteomics, Control, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Iron-Sulfur Clusters and Iron Responsive Element Binding Proteins Mediate Iron Accumulation in Corneal Endothelial Cells in Fuchs Dystrophy

doi: 10.1167/iovs.66.4.23

Figure Lengend Snippet: FDX1 knockdown increases susceptibility to RSL3-induced ferroptosis in immortalized human corneal endothelial cells (HCEC-B4G12). (A, B) Significant knockdown of FDX1 and ACO2 protein expression (Western blotting), respectively, regardless of siRNA sequences (i.e., siRNA-A, -B, or -C) against each mRNA target (see for bands). (C) MTS cell viability assay to demonstrate RSL3-induced ferroptosis in FDX1- or ACO2-knockdown HCEC-B4G12 cells. Ferrostatin-1 (Fer-1; 5 µM) acts as a ferroptosis inhibitor and RSL3 acts as a ferroptosis inducer. Data are represented as mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. (A, B) Violet-blue dots represent the siRNA control group and magenta dots represent the siRNA-mediated knockdown groups. (C) Violet-blue dots represent the siRNA control group, magenta dots represent the FDX1 knockdown group, and magenta triangles represent the ACO2 knockdown group.

Article Snippet: Immortalized human corneal endothelial cells (HCEC-B4G12; Leibniz Institute DSMZ, Braunschweig, Germany) were cultured in Opti-MEM I Reduced Serum Medium (Cat. no. 51985091; ThermoFisher) containing the following supplements: 5 ng/mL human epidermal growth factor (Cat. no. E9644; Sigma-Aldrich Corp., St. Louis, MO, USA); 20 ng/mL mouse nerve growth factor 2.5S (Cat. no. N6009; Sigma-Aldrich Corp.); 50 μg/mL gentamicin (Cat. no. IBI02030; IBI Scientific, Dubuque, IA, USA); 100 μg/mL Normocin (Cat. no. ant-nr-1; InvivoGen, San Diego, CA, USA); 0.8 mg/mL chondroitin sulfate (provided by Numedis, Inc, Isanti, MN, USA); 200 mg/L calcium chloride (Cat. no. C7902; Sigma-Aldrich); and 8% fetal bovine serum (Cat. no. S11150; R&D Systems, Flowery Branch, GA, USA).

Techniques: Knockdown, Expressing, Western Blot, Viability Assay, Control

Journal: Cell Reports Medicine

Article Title: A first-in-human clinical study of an allogenic iPSC-derived corneal endothelial cell substitute transplantation for bullous keratopathy

doi: 10.1016/j.xcrm.2024.101847

Figure Lengend Snippet:

Article Snippet: Human: HCEC-B4G12 , Leibniz Institute DSMZ , ACC 647.

Techniques: Recombinant, Injection, Mutagenesis, Control, Software, Microscopy, Digital PCR

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD primary and immortalized cell cultures show key marker s of ferroptosis. (A) TFR1 mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. ( B ) FSP1 mRNA expression in primary cells. ( C ) GPX4 mRNA expression in human expanded TCF4 repeat expansion primary cells. (D) FTH mRNA expression in non-FECD and FECD donor expanded TCF4 repeat expansion primary cells. (E) FTL mRNA expression in non-FECD and FECD donor primary cells. (F) Representation of median of the fluorescence of DHE showing significant difference in ROS between indicated cells. DHE (FL2 fluorescence) peak of F35T cells shifts to right when compared to B4G12 cells. (G) Representative confocal images showing fluorescence of DHE indicating ROS in the indicated cell lines. (H) Mitochondrial ROS quantified by MitoROS 580 dye in the indicated cells. Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, one-way ANOVA, followed by Tukey's post-hoc test. AMA indicates antimycin-A . (I) GPX4 mRNA and protein expression in HCEC-B4G12 and F35T cells. ( J ) Basal level of lipid peroxidation in HCEC-B4G12 and F35T cells quantified by C11-BODIPY fluorescent probe using flow cytometry. Comparisons of median fluorescence of C11-BODIPY detected in HCEC-B4G12 and F35T cells (10,000 cells). Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, Student's t-test. C11-BODIPY (FL1 fluorescence) peak of F35T cells shifts to right when compares to B4G12 cells. ( K ) Representative confocal images showing fluorescence of reduced and oxidized dye in the indicated cell lines. ( L ) 4-HNE protein expression in HCEC-B4G12 and F35T cells. All data of mRNA and protein expression are shown as mean ± SEM for n = 5–9 (B4G12), n = 5–7 (F35T) and n = 4 (both non-FECD and FECD donor primary cells). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗∗∗p < 0.001. Relative gene expression is normalized by β -actin.

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Marker, Expressing, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD immortalized cell culture show s key gene and protein expression changes related to ferroptosis. (A) TFR1 mRNA and protein expression in HCEC-B4G12 and F35T cells. (B) FTH mRNA expression in indicated cells. (C) FTL mRNA expression in indicated cells. ( D ) FSP1 mRNA and protein expression in the indicated cells. All data of mRNA and protein expression are shown as mean ± SEM for n = 5–9 (B4G12) and n = 5–7 (F35T). All the statistical comparisons were conducted using two-tailed, unpaired Student's t-test, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Relative gene expression is normalized by β-actin . ( E ) Representation of median of the fluorescence of FerroOrange showing significant difference in cytosolic Fe 2+ between indicated cells. B4G12 and F35T cells were stained with FerroOrange fluorescent probe to quantify cytosolic Fe 2+ by flow cytometry. A minimum 10,000 cells were quantified for measuring the fluorescence. (F) Cell population distribution after staining with FerroOrange showing cytosolic Fe 2+ content. (G) Representation of confocal microscopy images of HCEC-B4G12 and F35T cells stained with FerroOrange fluorescent probe showing and comparing cytosolic Fe 2+ . (H) Representation of median of the fluorescence of Mito-FerroGreen showing significant difference in mitochondrial Fe 2+ between indicated cells. HCEC-B4G12 and F35T cells were stained with Mito-FerroGreen fluorescent probe to quantify mitochondrial Fe 2+ by flow cytometry. A minimum of 10,000 cells were quantified for measuring fluorescence. (I) Cell population distribution after staining with Mito-FerroGreen showing mitochondrial Fe 2+ content. (J) Representation of confocal microscopy images of HCEC-B4G12 and F35T cells stained with Mito-FerroGreen fluorescent probe showing and comparing mitochondrial Fe 2+ . Data are represented as means ± SEM; n = 3; unpaired Student's t-test, ∗∗∗∗p < 0.0001. (K) Quantification of human ferritin (Ft) by ELISA in protein from HCEC-B4G12 and F35T cells. The human ferritin levels are presented as mean ± SEM; n = 9 (3 biological replicates × 3 technical replicates); ∗∗∗p < 0.001, Student's t-test. ( L) Quantification of mitochondrial ferritin (FtMt) by ELISA in protein from HCEC-B4G12 and F35T cells. The mitochondrial ferritin levels are presented as mean ± SEM; n = 9 (3 biological replicates × 3 technical replicates); ∗∗∗∗p < 0.0001, Student's t-test. (M) Representation of cellular iron metabolism in ferroptosis process.

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Cell Culture, Expressing, Two Tailed Test, Fluorescence, Staining, Flow Cytometry, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD demonstrates higher lipid droplets and higher susceptibility to ferroptosis induced by RSL3. (A) Representation of median fluorescence of LipidSpot™ 610 showing significant difference in ROS between indicated cell lines. LipidSpot™ 610 (FL4 fluorescence) peak of F35T cells shifts to right when compared to B4G12 cells. (B) Representative confocal images showing fluorescence of LipidSpot™ 610 indicating lipid droplets in the indicated cell lines. In flow cytometer analysis, data are shown as mean ± SEM; n = 9 (3 biological replicates × 3 technical replicates); ∗∗∗∗p < 0.0001, Student's t-test. (C) Representative confocal images showing RSL3 induced lipid droplets in the indicated cells. ( D ) Time lapse confocal images of ferroptosis events induced by RSL3. (E) Deferoxamine (DFO) iron chelation assay where HCEC-B4G12 and F35T cells were treated with DFO for 24h and then challenged with RSL3 at 1, 2 and 5 μM for different durations of 2, 4, 6 and 8h. Cell viability was measured by MTS assay. Data are represented as mean ± SEM for three biological replicates; Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Fluorescence, Flow Cytometry, MTS Assay

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: FECD demonstrates higher iron overload and higher susceptibility to ferroptosis induced by UVA irradiation. (A) Cytosolic Fe 2+ release in human donor corneas upon UVA irradiation at 5 J/cm 2 . Primary endothelial cells were isolated after UVA irradiation and stained with FerroOrange fluorescent probe. The experiment was conducted pairwise, where the left cornea was used as control and the right cornea was exposed to UVA. Data are shown as geometric mean FerroOrange (FL2 fluorescence) signals; n = 6 paired biological samples. (B) Comparison of geometric mean of FerroOrange (FL2 fluorescence) signals. Data are mean ± SEM, Paired two-tailed Student's t-test and n = 6 paired biological samples. (C) Representation of percent increase of labile cytosolic Fe 2+ after UVA irradiation at the fluence of 1, 2, 4 and 8 J/cm 2 . Indicated cells were stained with FerroOrange fluorescent probe immediately post-UVA (Data are shown as mean ± SEM, n = 9; 3 biological replicates × 3 technical replicates; ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, one-way ANOVA, followed by Tukey's post-hoc test). (D) Representation of percent increase of mitochondrial Fe 2+ after UVA irradiation at the fluence of 1, 2, 4 and 8 J/cm 2 . Indicated cells were stained with Mito-FerroGreen fluorescent probe immediately post-UVA (Data are shown as mean ± SEM, n = 9; 3 biological replicates × 3 technical replicates; ∗∗∗∗p < 0.0001, one-way ANOVA, followed by Tukey's post-hoc test.). ( E ) Time lapse confocal images of ferroptosis events induced by UVA irradiation at 1.5 J/cm 2 . Representative confocal images showing disappearance of lipid droplets at the time of ferroptosis induced death in the indicated cells. SYTOX green indicates cell death. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Irradiation, Isolation, Staining, Control, Fluorescence, Comparison, Two Tailed Test

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: Solubilized ubiquinol gives protection against ferroptosis. (A) Cell viability assay was performed by quantifying LDH release after treatment with solubilized ubiquinol at different concentrations of 1, 5, 10, 50, and 100 μM. Following solubilized ubiquinol treatment, HCEC-B4G12 and F35T cells were challenged with 1 μM of RSL3 for 24 h. Data are shown as mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, Student's t-test against RSL3 alone group. (B) Comparisons of cell viability of HCEC-B4G12 and F35T cells following the treatment of solubilized ubiquinol. Solubilized ubiquinol was comparatively less effective in F35T cells, indicating more ferroptosis compared to B4G12 cells. Data are shown as mean ± SEM; n = 3; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, Student's t-test. (C) Representative light microscopy images showing that solubilized ubiquinol at 1 μM dose inhibited RSL3 induced ferroptosis in HCEC-B4G12 and F35T cells. (D) Solubilized ubiquinol outperforms NAC, DFO and ferrostatin-1 in protecting F35T cells against RSL3 induced cell death in ferroptosis assay. Data are shown as mean ± SEM, n = 3; ∗∗∗∗p < 0.0001, Student's t-test.

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Viability Assay, Light Microscopy

Journal: Redox Biology

Article Title: TCF4 trinucleotide repeat expansions and UV irradiation increase susceptibility to ferroptosis in Fuchs endothelial corneal dystrophy

doi: 10.1016/j.redox.2024.103348

Figure Lengend Snippet: Solubilized ubiquinol gives protection against lipid peroxidation. (A) Schematic showing RSL3 induced ferroptosis and role of solubilized ubiquinol to prevent lipid peroxidation and ferroptosis. (B) Solubilized ubiquinol prevents lipid peroxidation in HCEC-B4G12 and F35T cells induced by RSL3 in dose dependent manner detected by the peak shift of C11-BODPY fluorescent probe in flow cytometer analysis. (C) C11-BODIPY fluorescence signals detected by flow cytometer following treatments with solubilized ubiquinol at different concentrations of 1, 10, 50 and 100 μM. Values are mean ± SEM; n = 3; ∗∗∗∗p < 0.0001, Student's t-test against RSL3-untreated group. (D) Representation of cell population undergoing RSL3 induced lipid peroxidation following solubilized ubiquinol treatment at the indicated concentrations in HCEC-B4G12 and F35T cells. Solubilized ubiquinol decreased the number of cells undergoing lipid peroxidation in a concentration dependent manner. Values are mean ± SEM; n = 3.

Article Snippet: Healthy immortalized human corneal endothelial cells (HCEC-B4G12) were procured from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture GmbH, Germany, and FECD immortalized human corneal endothelial cells (F35T) were a generous gift of Dr. Albert Jun (Johns Hopkins University, Baltimore, MD).

Techniques: Flow Cytometry, Fluorescence, Concentration Assay