cyppa  (Alomone Labs)

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 2. Intracellular calcium levels are increased by NIST DEP and RSL-3 co-treatment. (A) Fluo4-AM fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Green: Fluo4-AM positive cells, Blue: nucleus (scale bar =100 μm). (B) Quantitative analysis of Fluo4-AM positive cells was performed based on 10 fluorescent images per condition (mean ± SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 3. ER-mitochondria contact sites are increased by NIST DEP and RSL-3 co-treatment. (A) PLA fluorescence images at 40x magnification after 6 hrs exposure to NIST DEP and RSL-3 with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: PLA dots, Blue: nucleus (scale bar = 10 μm). (B) Quantification of PLA dots from 50 single cells based on fluorescence images for each condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 4. Mitochondrial calcium levels are increased by NIST DEP and RSL-3 co-treatment. (A) Mt-fura-2 fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Green: Fura 340, Blue: Fura 380 (scale bar = 20 μm). The ratio of fluorescence measured upon excitation at 340/380 nm (F340/F380) is shown in the right panel, ratio range from 0 to 2. (B) Fura340/Fura380 ratio picture of different treatments. Enlarged images of the boxed area were shown in the right panels. (C) Quantitative analysis of F340/F380 was performed based on 10 fluorescent images per condition (mean ± SD). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ***p < 0.001, ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 5. HT22 ferroptotic cell death induced by RSL-3 and NIST DEP is alleviated by inhibition of mitochondrial calcium uptake. (A) PI/Hoechst 33342 double-stained fluorescence images at 40x magnification after 17 hrs exposure to NIST DEP, RSL-3, calcium regulators MCUi4 (5 μM) and CyPPA (10 μM) alone or in combination. Red: dead cells, Blue: nucleus (scale bar =100 μm). (B) Quantitative analysis of PI-positive cells was performed based on 10 fluorescent images per condition (mean ± SD). The percentage of dead cells in the control group was defined as 100 %. (C) MTT assay for the viability of HT22 cells after 17 hrs exposure to NIST DEP, RSL-3, calcium regulators MCUi4 (5 μM) and CyPPA (10 μM) alone or in combination. The cell viability of the control group was defined as 100 %. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Inhibition, Staining, Fluorescence, Control, MTT Assay, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 6. Co-exposure to RSL-3 and NIST DEP induce mitochondrial morphological alteration. (A) Mitotracker Red fluorescence images at 63x magnification after 17 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: mito chondrial, Blue: nucleus (scale bar = 20 μm). Enlarged images of the boxed area are shown in the right panels. (B) Mitochondrial skeleton images at 63x magni fication after 17 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). (C-D) Parameters of HT22 mitochondrial network morphology analysis after different treatments: (C) Mitochondrial footprint. 15 single cells were analyzed for each condition. (D) Mean branch length. 10 whole fluorescence images were analyzed for each condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Comparison

Journal: Journal of hazardous materials

Article Title: Regulation of calcium signaling prevents neuronal death mediated by NIST DEP in xenoferroptotic cell death conditions.

doi: 10.1016/j.jhazmat.2025.137374

Figure Lengend Snippet: Fig. 7. Lipid peroxidation induced by RSL-3 and NIST DEP can be alleviated by MCUi4 and CyPPA. (A) BODIPY 581/591 C11fluorescence images at 20x magnification after 6 hrs exposure to NIST DEP and RSL-3 alone and in combination with or without the calcium regulators MCUi4 (5 μM) and CyPPA (10 μM). Red: C11non-ox, Green: C11ox, Blue: nucleus (scale bar = 100 μm). (B) Quantitative analysis of BODIPY 581/591 C11 mean fluorescence intensity was performed based on 10 fluorescent images per condition (mean ± SD). (C) MDA levels after 6 hrs of different treatments. All conditions were normalized by their corresponding protein concentrations, and the MDA concentration of the control group was defined as 1. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple comparison test. ***p < 0.001, ****p < 0.0001. All experiments were repeated at least three times as biological replicates.

Article Snippet: Ferroptosis inducer RSL-3 (#S8155, Selleckchem, USA), specific ferroptosis inhibitor Ferrostatin-1 (Fer-1; #SML0583, Sigma-Aldrich, Germany), apoptosis inhibitor QVD (#1135695–98–5, MP Biomedicine, USA), mitochondrial L. Zhang et al. Journal of Hazardous Materials 488 (2025) 137374 calcium uniporter (MCU) inhibitor MCUi4 (#7195, Tocris bioscience, UK) and small conductance calcium-activated potassium (SK) channel activator CyPPA (#73029–73–9, Tocris bioscience, UK) were added to HT22 cells for co-treatment.

Techniques: Fluorescence, Concentration Assay, Control, Comparison