mouse anti aif  (Santa Cruz Biotechnology)

Journal: Cell Death & Disease

Article Title: Transferrin is a drug candidate for the treatment of dry age-related macular degeneration (AMD)

doi: 10.1038/s41419-025-07950-0

Figure Lengend Snippet: A A 24-h incubation with 120 µM 4-hydroxy-2-nonenal (4HNE) significantly reduced human induced pluripotent stem cell (iPSC)-derived RPE (iRPE) cell viability compared with that of the control. Co-treatment with 10 mg/mL TF significantly protected iRPE cells from 4HNE-induced toxicity ( n = 8 wells per condition). B Transepithelial electrical resistance (TEER) was measured to evaluate iRPE cell integrity. TF co-treatment significantly prevented the decrease in TEER caused by 4HNE stress ( n = 11–12 wells per condition). C Apoptotic-induced factor 1 (AIF1) immunofluorescence-labeled mitochondria in iRPE cells treated with 4HNE or co-treated with TF (scale bar: 100 µm) were quantified ( D ), revealing the preservation of AIF1 staining when TF was added to 4HNE ( n = 4 wells per condition). E Under 4HNE stress, membrane integrity, monitored by the release of lactate dehydrogenase (LDH), was lost, and TF co-treatment significantly prevented this loss ( n = 8 wells per condition). Untreated cells were used as control. Bars were means ± SEM. One-way ANOVA with post-hoc Bonferroni test ( A , E ) or Kruskal‒Wallis test with Dunn’s post-hoc test ( B , D ); NS non-significant ## p < 0.01, #### p < 0.0001 compared with the control; * p < 0.05, *** p < 0.001 compared with the 4HNE stress.

Article Snippet: iRPE cells were fixed in 4% paraformaldehyde (PAF, Inland Europe, Conflans sur Lanterne, France) for 20 min. Flat-mounted Transwells or cryosections of snap-frozen iRPE cells in OCT (Tissue Tek, Siemens Medical, Puteaux, France) were permeabilized and blocked respectively in 1% Triton X-100 (Merck) in PBS and 1% bovine serum albumin (BSA; Merck)/0.1% Triton in PBS, followed by overnight incubation with goat anti-rabbit ZO1 (1:200; 40-2200, Invitrogen, France), goat anti-mouse NLRP3 (1:100; ALX-804-819, Enzo Life Sciences, Farmingdale, NY, USA), goat anti-mouse C5b9 (1:50; sc-58935, Santa Cruz Biotechnologies, Heidelberg, Germany), goat anti-mouse AIF1 (1:50; sc-13116, Santa Cruz Biotechnologies), goat anti-rabbit 4HNE (1:100; ab46545, Abcam, Cambridge, UK) and goat anti-rabbit NRF2 (1:100; sc-722, Santa Cruz Biotechnologies) primary antibodies.

Techniques: Incubation, Derivative Assay, Control, Immunofluorescence, Labeling, Preserving, Staining, Membrane

Journal: Cell Death & Disease

Article Title: Transferrin is a drug candidate for the treatment of dry age-related macular degeneration (AMD)

doi: 10.1038/s41419-025-07950-0

Figure Lengend Snippet: A Sustained iron overload (0.5 mM FeCl 3 NTA for 48 h) induced a significant decrease in iRPE cell integrity, and TF (10 mg/mL) added at 24 h, restored the TEER to the control level ( n = 4 wells per condition). Mitochondrial viability was quantified via Apoptotic-induced factor 1 (AIF1) immunofluorescence labeling ( B ), which revealed a decrease in AIF1 staining after 48 h of FeCl 3 exposure ( C ). TF treatment preserved AIF1 staining. ( n = 4 wells per condition). D , E Perl’s reaction revealed intracellular iron accumulation in iRPE cells exposed to FeCl 3 for 48 h. TF added at the half-time of iron exposure decreased iron accumulation ( n = 4 wells per condition). F Transferrin receptor 1 (TFR1), ferritin light chain ( FTL), and ferroportin (FPN) immunoblotting revealed an iron homeostasis perturbation by sustained iron overload in iRPE cells. The TF treatment restored the protein level to the control level ( n = 5–6 wells per condition). Sustained iron overload-induced lipid peroxidation, as evaluated by 4HNE immunofluorescence labeling ( G ) and quantified ( H ) after 48 h. TF treatment significantly reduced 4HNE staining compared to FeCl 3 treatment ( n = 4 wells per condition). I Sustained iron overload in iRPE cells increased the expression of the ferroptosis-related genes heme oxygenase 1 ( HMOX1 ), solute carrier family 7 member 1 ( SLC7A11 ), cyclooxygenase 2 ( COX-2 ), acyl-CoA synthetase long chain family member 4 (ACSL4 ), and nuclear receptor coactivator 4 ( NCOA4 ). TF treatment prevented the expression of all the genes ( n = 6 wells per condition). J The glutathione peroxidase 4 (GPX4) protein levels, as quantified by immunoblotting was decreased by sustained iron overload but restored by TF treatment ( n = 5‒6 wells per condition). Untreated cells were used as control. Bars were means ± SEM. One-way ANOVA with post-hoc Bonferroni test ( F , I , J ) or Kruskal‒Wallis test with Dunn’s post-hoc test ( A , C , E , H ); NS non-significant, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with the control; ns non-significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with FeCl 3 . Scale bar: 100 µm.

Article Snippet: iRPE cells were fixed in 4% paraformaldehyde (PAF, Inland Europe, Conflans sur Lanterne, France) for 20 min. Flat-mounted Transwells or cryosections of snap-frozen iRPE cells in OCT (Tissue Tek, Siemens Medical, Puteaux, France) were permeabilized and blocked respectively in 1% Triton X-100 (Merck) in PBS and 1% bovine serum albumin (BSA; Merck)/0.1% Triton in PBS, followed by overnight incubation with goat anti-rabbit ZO1 (1:200; 40-2200, Invitrogen, France), goat anti-mouse NLRP3 (1:100; ALX-804-819, Enzo Life Sciences, Farmingdale, NY, USA), goat anti-mouse C5b9 (1:50; sc-58935, Santa Cruz Biotechnologies, Heidelberg, Germany), goat anti-mouse AIF1 (1:50; sc-13116, Santa Cruz Biotechnologies), goat anti-rabbit 4HNE (1:100; ab46545, Abcam, Cambridge, UK) and goat anti-rabbit NRF2 (1:100; sc-722, Santa Cruz Biotechnologies) primary antibodies.

Techniques: Control, Immunofluorescence, Labeling, Staining, Western Blot, Expressing