human retinal pigment epithelium (hrpe) cells  (Novogenix Laboratories)

Journal: bioRxiv

Article Title: Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

doi: 10.1101/2025.06.19.660627

Figure Lengend Snippet: (A) hRPE cells were treated with 200 µg/ml for 24 and 48 hr in the absence or presence of PMC (1.3 µM). Cytotoxicity was also measured in untreated (control) and PMC alone treated cells. Cell death was measured in the condition media using the LDH assay, n=3. Values are expressed as mean ± SEM. Statistical analysis was conducted using one-way ANOVA, ***P<0.001, n=3. Heatmap of top differently expressed genes of bulk RNA sequencing in (B) apoptosis and (c) antioxidant pathways, n=3. Colors shows intensity in z-scored units where red shows replicates with high expression (z-score= +5) and blue shows replicates with low expression (z-score =-5).

Article Snippet: Primary human fetal retinal pigmented epithelium cells (hRPE) (Lonza, USA) were cultured in 50% (v/v) DMEM/F12 (Life Technologies, USA)/50% (v/v) αMEM (Sigma-Aldrich, USA) supplemented with 1x penicillin-streptomycin, Glutamax, sodium pyruvate, non-essential amino acids (all from Life Technologies, USA).

Techniques: Control, Lactate Dehydrogenase Assay, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

doi: 10.1101/2025.06.19.660627

Figure Lengend Snippet: (A) hRPE cells matured for 4 weeks, were treated with ox-LDL (200 µg/ml) with or without the presence of PMC (1.3 µM) in serum-free media. Relative HMOX1 transcript levels were measured using qPCR. (B) Western blotting of the hRPE cells with the above indicated treatments at 24 and 48 hr was conducted to examine HO-1 levels. (C) Quantification was performed using densitometry after normalization with GAPDH, n=3. (D) HMOX-1 mRNA levels were determined by PCR in serum-starved ARPE-19 cells treated with ox-LDL in the presence or absence of PMC and qPCR. (E) Cell lysates from ARPE-19 cells treated as above-were examined using western blot to determined HO-1 levels. (F) Quantification of HO-1 levels were conducted after normalization with α -tubulin. HMOX1/HO-1 was also assessed in hRPE and ARPE-19 cells treated with PMC alone. Serum-starved untreated cells were considered as control for all the experiments. Values are indicated as mean ± SEM of n=3. One-way ANOVA was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001

Article Snippet: Primary human fetal retinal pigmented epithelium cells (hRPE) (Lonza, USA) were cultured in 50% (v/v) DMEM/F12 (Life Technologies, USA)/50% (v/v) αMEM (Sigma-Aldrich, USA) supplemented with 1x penicillin-streptomycin, Glutamax, sodium pyruvate, non-essential amino acids (all from Life Technologies, USA).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

doi: 10.1101/2025.06.19.660627

Figure Lengend Snippet: (A) Serum-starved hRPE cells were treated with ox-LDL (200 µg/ml) in the presence or absence of PMC (1.3 µM). NQO1 levels were determined using qPCR at 24 and 48 hr, n=6. (B) Western blot was conducted on cell lysates from hRPE cells treated with ox-LDL with or without PMC to determine NQO1 levels. (C) Densitometry was conducted with normalization for GAPDH, n=3. (D) Serum-starved ARPE-19 treated under the same experimental treatment conditions as above were analyzed for NQO1 levels using qPCR, n=6. (E) NQO1 protein levels were measured in cell lysates from ARPE-19 in the ox-LDL treated groups with or without PMC. (F) Densitometry analysis was conducted with normalization with GAPDH to quantify NQO1 levels, n=3. NQO1 levels were also measured in hRPE and ARPE-19 cells treated with PMC alone. Serum-starved untreated cells were considered as the control for all the experiments. Values are indicated as mean ± SEM of the indicated n. One-way ANOVA was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001

Article Snippet: Primary human fetal retinal pigmented epithelium cells (hRPE) (Lonza, USA) were cultured in 50% (v/v) DMEM/F12 (Life Technologies, USA)/50% (v/v) αMEM (Sigma-Aldrich, USA) supplemented with 1x penicillin-streptomycin, Glutamax, sodium pyruvate, non-essential amino acids (all from Life Technologies, USA).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

doi: 10.1101/2025.06.19.660627

Figure Lengend Snippet: (A) Illustration showing the timeline of 48 hr siHMOX1 silencing and 24 hr ox-LDL treatments in the presence or absence of PMC in hRPE cells. (B) Western blotting was conducted to estimate HO-1 levels in hRPE cells subjected to siScr or siHMOX1 for 48 hr followed by ox-LDL (200 µg/ml) treatments with or without PMC (1.3 µM). (C) Quantification of HO-1 levels was performed using densitometry after normalization with GAPDH, n=3. (D) LDH levels were measured in the conditioned media from siScr or siHMOX-treated cells along with the siScr and siHMOX1 cells that were treated with ox-LDL with or without PMC, n=14. (E) Illustration of the timeline indicating, 48 hr siHMOX1 silencing in hRPE cells followed by additional 48 hr treatment of ox-LDL with/without PMC. (F) HO-1 levels were detected in hRPE cells treated with siScr or siHMOX1 for 48 hr, followed by ox-LDL in the presence or absence of PMC using western blotting. (G) Densitometry analysis was conducted to quantify HO-1 levels after normalization with GAPDH, n=3. (H) Cytotoxicity was analyzed by assaying LDH in the conditioned media of siScr and siHMOX1 and siScr and siHMOX cells that were subjected to ox-LDL with/without PMC treatment, n=6. Cells treated with ox-LDL in the presence or absence of PMC without siScr or siHMOX1 were used as control for both 24 hr and 48 hr ox-LDL and ox-LDL with PMC treatments, n=6. Values are indicated as mean ± SEM of the indicated n. One-way ANOVA was used for statistical analysis, *P<0.05, **P<0.01, ***P<0.001

Article Snippet: Primary human fetal retinal pigmented epithelium cells (hRPE) (Lonza, USA) were cultured in 50% (v/v) DMEM/F12 (Life Technologies, USA)/50% (v/v) αMEM (Sigma-Aldrich, USA) supplemented with 1x penicillin-streptomycin, Glutamax, sodium pyruvate, non-essential amino acids (all from Life Technologies, USA).

Techniques: Western Blot, Control

Journal: bioRxiv

Article Title: Mechanism of PMC (2,2,5,7,8-pentamethyl-6-chromanol), a sterically hindered phenol antioxidant, in rescuing oxidized low-density lipoprotein-induced cytotoxicity in human retinal pigment epithelial cells

doi: 10.1101/2025.06.19.660627

Figure Lengend Snippet: Continuous PMC presence is required for protection against ox-LDL. (A) Serum-starved hRPE cells were treated with either ox-LDL (200 µg/ml) and PMC (1.3 µM) alone or, ox-LDL and PMC under different treatment conditions. ox-LDL+PMC denotes simultaneous treatment, while in other treatment groups either the cells were pretreated with PMC or with ox-LDL. Following these treatments, media were replaced with ox-LDL+PMC, ox-LDL alone, or PMC alone. In another group, media were not replaced, and treatments were added to the same media. Cells were pretreated with PMC and ox-LDL was added to the same media or pretreated with ox-LDL and an approximately 10-fold higher PMC concentration (10 µM) was added to the same media. Untreated cells were considered as controls. Values are indicated as mean ± SEM of n=3. One-way ANOVA was used for statistical analysis, ***P<0.001. (B) Graphical summary of the PMC mediated protection against ox-LDL in RPE. Uptake of ox-LDL via the CD36 receptor causes lysosomal destabilization and oxidative stress in RPE leading to ROS generation. This triggers Nrf2 dissociation from Keap1 and its translocation to the nucleus where it interacts with the specific promoter region, ARE, leading to the upregulation of HMOX1 and NQO1. PMC prevents this upregulation of HMOX1/HO-1 and NQO1 levels by preventing ROS generation. Created using BioRender.

Article Snippet: Primary human fetal retinal pigmented epithelium cells (hRPE) (Lonza, USA) were cultured in 50% (v/v) DMEM/F12 (Life Technologies, USA)/50% (v/v) αMEM (Sigma-Aldrich, USA) supplemented with 1x penicillin-streptomycin, Glutamax, sodium pyruvate, non-essential amino acids (all from Life Technologies, USA).

Techniques: Concentration Assay, Translocation Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) qRT-PCR analysis of NEAT1, SOX4 and miR-204 in ARPE19 cells at 12, 24, 48 and 72 h after high glucose induction. (B–C) SOX4 and EMT-associated proteins such as E-cadherin, N-cadherin, Snail, and Vimentin were examined by western blot (B) and cell immunofluorescence (C) at 12, 24, 48 and 72 h after high glucose induction, Scale bar: 50 µM. D. Cell migration was investigated by wound healing assay, the first row was the scratch size of control and high glucose treatment (12, 24, 48 and 72 h) group at 0 h, which was used as a baseline comparison, while the second row was the scratch size of corresponding groups at 72 h, which could be used to evaluate the treatment effects against high glucose by comparing with the first row. E. invasion ability was investigated by transwell assay E at 12, 24, 48 and 72 h after high glucose induction. Data were presented as the mean ± SD of three separated experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Migration, Wound Healing Assay, Control, Comparison, Transwell Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) ARPE19 cells was first transfected with si-NEAT1, then the expression level of NEAT1, SOX4 and miR-204 after 72 h high glucose induction were analyzed by qRT-PCR. (B) Cell viability were verified by MTT assay in experiments described in A. (C–D). SOX4 and EMT-associated proteins such as E-cadherin, N-cadherin, Snail, and Vimentin were examined by western blot (C) and cell immunofluorescence (D) after 72 h high glucose induction and 72 h si-NEAT1 transfection. Scale bar: 50 µM. (E–F). Cell migration and invasion ability was investigated by wound healing assay (E) and cell transwell assay (F). Data were presented as the mean ± SD of three separated experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Transfection, Expressing, Quantitative RT-PCR, MTT Assay, Western Blot, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) NEAT1, miR-204 and SOX4 in ARPE19 cells after 72 h high glucose induction and 72 h miR-204 mimic transfection was analyzed by qRT-PCR. (B) Cell viability was verified by MTT assay after 72 h high glucose induction and 72 h miR-204 mimic transfection. (C–D). SOX4 and EMT-associated proteins such as E-cadherin, N-cadherin, Snail, and Vimentin were examined by western blot (C) and cell immunofluorescence (D) after 72 h high glucose induction and 72 h miR-204 mimic transfection. Scale bar: 50 µM. (E–F). Cell migration and invasion ability was investigated by wound healing assay (E) and cell transwell assay (F) after 72 h high glucose induction and 72 h miR-204 mimic transfection. Data were presented as the mean ± SD of three separated experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Transfection, Quantitative RT-PCR, MTT Assay, Western Blot, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) ARPE19 cells were first transfected with si-NEAT1 or miR-204 mimics, then the expression level of SOX4 after 72 h high glucose induction were analyzed by qRT-PCR. (B) Putative binding site between miR-204 and NEAT1 or SOX4. (C) The luciferase activity was detected by a dual luciferase reporter system.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) SOX4 in ARPE19 cells after 72 h high glucose induction and 72 h si-SOX4 transfection was analyzed by qRT-PCR. (B–C). Cell viability and cell proliferation were verified by MTT assay(B) and BrdU assay (C) respectively after 72 h high glucose induction and 72 h si-SOX4 transfection. (D–E). SOX4 and EMT-associated proteins such as E-cadherin, N-cadherin, Snail, and Vimentin were examined by western blot (D) and cell immunofluorescence (E) after 72 h high glucose induction and 72 h si-SOX4 transfection. Scale bar: 50 µM. (F–G) Cell migration and invasion ability was investigated by wound healing assay (F) and cell transwell assay (G) after 72 h high glucose induction and 72 h si-SOX4 transfection. Data were presented as the mean ± SD of three separated experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Transfection, Quantitative RT-PCR, MTT Assay, BrdU Staining, Western Blot, Immunofluorescence, Migration, Wound Healing Assay, Transwell Assay

Journal: PeerJ

Article Title: LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis

doi: 10.7717/peerj.11817

Figure Lengend Snippet: (A) SOX4 in ARPE19 cells after 72 h high glucose induction and 72 h si-NEAT1 and miR-204 inhibitor co-transfection was analyzed by qRT-PCR. (B–C). Cell viability and cell proliferation were verified by MTT assay (B) and BrdU assay (C) respectively after 72 h high glucose induction and 72 h si-NEAT1 and miR-204 inhibitor co-transfection. (D) SOX4 and EMT-associated proteins such as E-cadherin, N-cadherin, Snail, and Vimentin were examined by western blot after 72 h high glucose induction and 72 h si-NEAT1 and miR-204 inhibitor co-transfection. (E–F) Cell migration and invasion ability was investigated by wound healing assay and cell transwell assay after 72 h high glucose induction and 72 h si-NEAT1 and miR-204 inhibitor co-transfection. Data were presented as the mean ± SD of three separated experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Human retinal pigment epithelium (hRPE) cell line ARPE19 was obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) with fetal bovine serum (FBS, 10%, Invitrogen, Carlsbad, CA, USA), L-glutamine (0.292 mg/mL; Invitrogen, Carlsbad, CA, USA) and penicillin (100 U/mL; Invitrogen, Carlsbad, CA, USA), and replaced every 3 days.

Techniques: Cotransfection, Quantitative RT-PCR, MTT Assay, BrdU Staining, Western Blot, Migration, Wound Healing Assay, Transwell Assay

Journal: Progress in retinal and eye research

Article Title: Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases

doi: 10.1016/j.preteyeres.2017.01.004

Figure Lengend Snippet: Status of registered clinical trials using cell-based therapies for AMD and RP.

Article Snippet: Astellas Institute for Regenerative Medicine , Advanced dry AMD , Ongoing, not recruiting , Safety and efficacy , Subretinal injection of human embryonic stem cell derived retinal pigment epithelium cells (MA09-hRPE) , USA (California, Florida, Massachusetts, Pennsylvania) , 10-Jun-16 , NCT02463344.

Techniques: Clinical Proteomics, Transplantation Assay, Derivative Assay, Injection, Membrane, Saline