hrpes  (ATCC)

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